Selected reaction monitoring assays

ABSTRACT

Provided herein are methods for developing selected reaction monitoring mass spectrometry (LC-SRM-MS) assays.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 14/738,146, filed Jun. 12, 2015, which is a continuation of U.S. application Ser. No. 13/725,098, filed Dec. 21, 2012, now U.S. Pat. No. 9,091,651, which claims priority and benefit of U.S. Provisional Application No. 61/578,718, filed Dec. 21, 2011, and U.S. Provisional Application No. 61/614,818, filed Mar. 23, 2012, the contents of each of which are incorporated herein by reference in their entireties.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “IDIA-004_C01US_ST25.txt”, which was created on Jun. 11, 2015 and is 395,000 bytes in size, are hereby incorporated by reference in their entireties.

BACKGROUND

Liquid Chromatography Selected Reaction Monitoring Mass Spectrometry (LC-SRM-MS) has emerged as an alternative technology to immunoassays for quantification of target proteins in biological samples. LC-SRM-MS methods are highly desirable because LC-SRM-MS methods provide both absolute structural specificity for the target protein and relative or absolute measurement of the target protein concentration when suitable internal standards are utilized. In contrast to immunoassays, LC-SRM-MS does not involve the manufacturing of biologics. LC-SRM-MS protein assays can be rapidly and inexpensively developed in contrast to the development of immunoassays. LC-SRM-MS are highly multiplexed, with simultaneous assays for hundreds of proteins performed in a single sample analysis. Using LC-SRM-MS in contrast to other proteomic technologies allows for complex assays for the identification diagnostic proteins in complex diseases such as cancer, autoimmune, and metabolic disease. In particular, the development of a highly multiplexed LC-SRM-MS assay that reproducibly identifies a specific set of proteins relevant to a clinical disease presents diagnostic advantages and efficiencies. To date, proteomic techniques have not enabled such inventions to exist where hundreds of proteins can be accurately quantified within a single sample. The present invention provides accurate measurement of hundreds of lung cancer associated proteins within a single sample using multiplexed techniques.

SUMMARY OF THE INVENTION

The present invention comprises a LC-SRM-MS assay for the measurement proteins in a single sample and in a single LC-SRM-MS assay. The assay was optimized for protein quantification and minimal interference among proteins in the assay. This LC-SRM-MS assay is novel because measurement of a large number of proteins in a single sample specifically associated with lung cancer has not been accomplished. Simultaneous measurement of such a large number of proteins without interference among the proteins requires specific techniques to distinguish among the proteins. The current invention provides clinical utility as this assay was used for development of lung cancer diagnostic tests for the early detection of lung cancer, managing disease treatment, as well as testing for disease recurrence.

The object of the present invention is to provide improved methods for the use of LC-SRM-MS in the development of assays. Accordingly, provided herein is a method for developing peptides and transitions for a plurality of at least 200 proteins for a single sample selected reaction monitoring mass spectrometry (LC-SRM-MS) assay, including the steps of providing a set of 200 or more proteins; generating transitions for each protein; assessing LC-SRM-MS data by Mascot score; performing collision energy optimization on the transitions; selecting peptides with transitions showing the greatest peak areas of their transitions; selecting a set of transitions for each peptide, wherein the transitions for each peptide have one of the four most intense b or y transition ions; the transitions for each peptide have m/z values of at least 30 m/z above or below those of the precursor ion; the transitions for each peptide do not interfere with transitions from other peptides; and the transitions represent transitions due to breakage of peptide bond at different sites of the protein.

In one embodiment of the method, each selected peptide in the set of peptides has a monoisotopic mass of 700-5000 Da; and does not contain a cysteine or a methionine; or may contain cysteine or methionine. In another embodiment, the transitions for each peptide have one of the four most intense b or y transition ions; have m/z values of at least 30 m/z above or below those of a precursor ion; do not interfere with transitions from other peptides; and represent transitions due to breakage of peptide bond at different sites of the protein.

In another embodiment of the method, the peptides do not include any peptide that is bounded by KK, KR, RK or RR (either upstream or downstream) in the corresponding protein sequence. Specifically, the amino acid is charged at pH 7.0. These amino acids include arginine and lysine. In another embodiment, each peptide of said set of peptides is unique to the corresponding protein. In yet another embodiment, the peptides do not include peptides which were observed in post-translational modified forms. In still another embodiment, each set of peptides is prioritized according to one or more of the following ordered set of criteria: unique peptides first, then non-unique; peptides with no observed post-translational modifications first, then those observed with post-translational modifications; peptides within the mass range 800-3500 Da first, then those outside of 800-3500 Da; and sorted by decreasing number of variant residues. In certain embodiments, the peptides are unique in that they only appear once among the peptides run in a single assay.

In one embodiment, each set of peptides is prioritized according to all of the ordered set of criteria. In another embodiment, each prioritized set of peptides contains 1-5 peptides.

In certain embodiments of the preceding methods, the two best peptides per protein and the two best transitions per peptide are selected based on experimental data resulting from LC-SRM-MS analysis of one or more of the following experimental samples: a biological disease sample, a biological control sample, and a mixture of synthetic peptides of interest. In a particular embodiment, the biological disease and biological control samples are processed using an immunodepletion method prior to LC-SRM-MS analysis. In another embodiment, the experimental samples contain internal standard peptides. In yet another embodiment, the LC-SRM-MS analysis method specifies a maximum of 7000 transitions, including transitions of the internal standard peptides and transitions. In other embodiments the method specifies a maximum of between 1000-7000, 2000-6000, 3000-5000 and about 3500 transitions.

In one embodiment of the method, the top two transitions per peptide are selected according to one or more of the following criteria the transitions exhibit the largest peak areas measured in either of the two biological experimental samples; the transitions are not interfered with by other ions; the transitions do not exhibit an elution profile that visually differs from those of other transitions of the same peptide; or the transitions are not beyond the detection limit of both of the two biological experimental samples.

In another embodiment of the method, the top two peptides per protein are selected according to one or more of the following criteria: one or more peptides exhibit two transitions and represent the largest combined peak areas of the two transitions; or one or more peptides exhibit one transition and represent the largest combined peak areas of the two transitions.

In another aspect, provided herein is an assay developed according to the foregoing method, and embodiments thereof.

In yet another aspect provided herein is the use of an assay developed according to the foregoing method, and embodiments thereof, to detect a plurality of at least 200 proteins in a single biological sample.

In another aspect, provided herein is an assay developed according to the foregoing method, and embodiments thereof.

The disclosure provides a composition comprising at least five transition ions selected from the listing of transition ions in Table 2. In one embodiment of the assay each transition ion independently corresponds to a unique protein. The five transition ions corresponded to proteins selected from the group consisting of LRP1, BGH3, COIA1, TETN, TSP1, ALDOA, GRP78, ISLR, FRIL, LG3BP, PRDX1, FIBA, and GSLG1. (see: U.S. application Ser. No. 13/306,823 PCT/US11/62461). The composition can further include an additional five transition ions selected from the listing of transition ions in Table 2. The additional five transition ions can corresponded to the proteins APOE, BASP1, CD14, FOXA2 and HSPB1.

The disclosure provides a composition comprising at least five synthetic peptides selected from the listing of peptides and proteins in Table 2. In one embodiment, each peptide can independently correspond to a unique protein. At least one of the peptides was isotopically labeled. The amount of each of the at least five synthetic peptides is known. In another embodiment, the composition included one or more polar solvents. The five synthetic peptides can correspond to the proteins LRP1, BGH3, COIA1, TETN, TSP1, ALDOA, GRP78, ISLR, FRIL, LG3BP, PRDX1, FIBA, and GSLG1. The composition can also include an additional five synthetic peptides selected from the listing of peptides and proteins in Table 2. The additional five synthetic peptides can correspond to the proteins APOE, BASP1, CD14, FOXA2 and HSPB1.

The disclosure provides a use of a composition, as described above, for the development of an assay to detect a disease, disorder or condition in a mammal.

The disclosure provides a method comprising analyzing a composition, as described above, using mass spectrometry. The method can use selected reaction monitoring mass spectrometry.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts candidate protein cohort by source. 217 tissue proteins were identified using proteomics analysis. 319 proteins were identified by review of the literature. Between the two sources, there was an overlap of 148 proteins.

FIG. 2 is a bar diagram showing Pearson correlations for peptides from the same peptide, from the same protein and from different proteins.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure relates to methods for developing peptides and transitions for a single sample selected reaction monitoring mass spectrometry (LC-SRM-MS) assay, generally comprising the steps of providing a set of proteins; identifying representative proteolytic peptides for each protein according to a set of criteria; identifying representative transitions for each peptide according to another set of criteria; and selecting the optimum peptides per protein and the optimum transitions per peptide.

Selected reaction monitoring mass spectrometry is capable of highly sensitive and accurate protein quantification based on the quantification of proteolytic peptides. In terms of clinical utility, mass spectrometry-based assays are often compared to immunoassays (e.g., Enzyme-Linked Immunosorbent Assay, or ELISA), which have the ability to quantify specific analytes in large sample sets (e.g., 96 or 384 samples in parallel microtitre plate-based format). Until recently, mass spectrometry-based protein assays were not able to match these sample sizes or quantitative accuracy. Considerable time and expense is required to generate and characterize antibodies required for immunoassays. Increasingly efficient LC-SRM-MS assays, therefore, may surpass immunoassays such as ELISA in the rapid development of clinically useful, multiplexed protein assays.

LC-SRM-MS is a highly selective method of tandem mass spectrometry which has the potential to effectively filter out all molecules and contaminants except the desired analyte(s). This is particularly beneficial if the analysis sample is a complex mixture which may comprise several isobaric species within a defined analytical window. LC-SRM-MS methods may utilize a triple quadrupole mass spectrometer which, as is known in the art, includes three quadrupole rod sets. A first stage of mass selection is performed in the first quadrupole rod set, and the selectively transmitted ions are fragmented in the second quadrupole rod set. The resultant transition (product) ions are conveyed to the third quadrupole rod set, which performs a second stage of mass selection. The product ions transmitted through the third quadrupole rod set are measured by a detector, which generates a signal representative of the numbers of selectively transmitted product ions. The RF and DC potentials applied to the first and third quadrupoles are tuned to select (respectively) precursor and product ions that have m/z values lying within narrow specified ranges. By specifying the appropriate transitions (m/z values of precursor and product ions), a peptide corresponding to a targeted protein may be measured with high degrees of sensitivity and selectivity. Signal-to-noise ratio in LC-SRM-MS is often superior to conventional tandem mass spectrometry (MS/MS) experiments that do not selectively target (filter) particular analytes but rather aim to survey all analytes in the sample.

Accordingly, provided herein is a method for developing peptides and transitions for a plurality of proteins for use in selected reaction monitoring mass spectrometry (LC-SRM-MS) assay. In a preferred embodiment, the assay involves the analysis of a single sample containing all analytes of interest (e.g., a proteolytic digest of plasma proteins). As to the selection of the protease(s) used, trypsin, which cleaves exclusively C-terminal to arginine and lysine residues, is a preferred choice to generate peptides because the masses of generated peptides are compatible with the detection ability of most mass spectrometers (up to 2000 m/z), the number and average length of generated peptides, and also the availability of efficient algorithms for the generation of databases of theoretical trypsin-generated peptides. High cleavage specificity, availability, and cost are other advantages of trypsin. Other suitable proteases will be known to those of skill in the art. Miscleavage is a factor for failure or ambiguous protein identification. A miscleavage can be defined as partial enzymatic protein cleavages generating peptides with internal missed cleavage sites reflecting the allowed number of sites (targeted amino acids) per peptide that were not cut. The presence of post-translational modifications (PTMs) is also a potential contributor to the problem of miscleavages.

LC-SRM-MS mass spectrometry involves the fragmentation of gas phase ions and occurs between the different stages of mass analysis. There are many methods used to fragment the ions and these can result in different types of fragmentation and thus different information about the structure and composition of the molecule. The transition ions observed in an LC-SRM-MS spectrum result from several different factors, which include, but are not limited to, the primary sequence, the amount of internal energy, the means of introducing the energy, and charge state. Transitions must carry at least one charge to be detected. An ion is categorized as either a, b or c if the charge is on a transition comprising the original N terminus of the peptide, whereas the ion is categorized as either x, y or z if the charge is on a transition comprising the original C terminus of the peptide. A subscript indicates the number of residues in the transition (e.g., one peptide residue in x₁, two peptide residues in y₂, and three peptide residues in z₃, etc.).

In a generic peptide repeat unit represented —N—C(O)—C—, an x ion and an a ion resulting from cleavage of the carbonyl-carbon bond (i.e., C(O)—C). The x ion is an acylium ion, and the a ion is an iminium ion. A y ion and a b ion result from cleavage of the carbonyl-nitrogen bond (i.e., C(O)—N, also known as the amide bond). In this case, the y ion is an ammonium ion and the b ion is an acylium ion. Finally, a z ion and a c ion result from cleavage of the nitrogen-carbon (i.e., C—N) bond. The z ion is a carbocation and the c ion is an ammonium ion.

Superscripts are sometimes used to indicate neutral losses in addition to the backbone fragmentation, for example, * for loss of ammonia and ° for loss of water. In addition to protons, c ions and y ions may abstract an additional proton from the precursor peptide. In electrospray ionization, tryptic peptides may carry more than one charge.

Internal transitions arise from double backbone cleavage. These may be formed by a combination of b-type and y-type cleavage (i.e., cleavage producing b and y ions). Internal cleavage ions may also be formed by a combination of a-type and y-type cleavage. An internal transition with a single side chain formed by a combination of a-type and y-type cleavage is called an iminium ion (sometimes also referred to as an imonium or immonium ion). These ions are labeled with the one letter code for the corresponding amino acid.

Low energy CID (i.e., collision induced dissociation in a triple quadrupole or an ion trap) involves the fragmentation of a peptide carrying a positive charge, primarily along its backbone, to generate primarily a, b and y ions.

In one aspect, provided herein is a method for developing peptides and transitions for a plurality of proteins for a single sample selected reaction monitoring mass spectrometry (LC-SRM-MS) assay, by: (a) providing a panel of a plurality of proteins; (b) identifying a set of peptides for each protein, wherein (i) each peptide in the set of peptides corresponds to a transition of said protein; (ii) the peptides have a monoisotopic mass of 700-5000 Da; and (iii) the peptides do not contain a cysteine or a methionine; or may contain cysteine or methionine; (c) identifying a set of transitions for each peptide, wherein (i) the transitions for each peptide have one of the four most intense b or y transition ions; (ii) the transitions for each peptide have m/z values of at least 30 m/z above or below those of the precursor ion; (iii) the transitions for each peptide do not interfere with transitions from other peptides; and (iv) the transitions represent transitions due to breakage of peptide bond at different sites of the protein; and (d) selecting the peptides for each protein that best fit the criteria of step (b) and the transitions per peptide that best fit the criteria of step (c); thereby developing peptides and transitions for a LC-SRM-MS assay.

By plurality of proteins it is meant at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more In certain embodiments, the plurality of proteins can encompass between 2 and 10. 10 and 20, 20 and 50, 50 and 100, 100 and 200, or 200 and 500 proteins. In other embodiments, the plurality of proteins can encompass between 250 and 450; or 300 and 400 proteins.

Trypsin-like proteases cleave peptide bonds following a positively charged amino acid (e.g., lysine (K) or arginine (R)). This specificity is driven by the residue which lies at the base of the enzyme's S1 pocket (generally a negatively charged aspartic acid or glutamic acid). Accordingly, in one embodiment of the method, the peptides do not include any peptide that is bounded by KK, KR, RK or RR, either upstream of downstream in the corresponding protein sequence. In another embodiment, each peptide of said set of peptides is unique to the corresponding protein.

Post-translational modification (PTM) is the chemical modification of a protein after its translation. It can include any modification following translation, including cleavage. It is one of the later steps in protein biosynthesis, and thus gene expression, for many proteins. It is desirable to avoid such peptides for the purpose of protein identification. Thus, in another embodiment, the peptides do not include peptides which were observed in post-translational modified forms.

In still another embodiment, each set of peptides is prioritized according to one or more of the following ordered set of criteria: (a) unique peptides first, then non-unique; (b) peptides with no observed post-translational modifications first, then those observed with post-translational modifications; (c) peptides within the mass range 800-3500 Da first, then those outside of 800-3500 Da; and (d) sorted by decreasing number of variant residues. In one embodiment, each set of peptides is prioritized according to all of the ordered set of criteria. In another embodiment, each prioritized set of peptides contains 1-5 peptides.

In certain embodiments, one or more liquid chromatography (LC) purification steps are performed prior to a subsequent LC-SRM-MS analysis step. Traditional LC analysis relies on the chemical interactions between sample components and column packing materials, where laminar flow of the sample through the column is the basis for separation of the analyte of interest from the test sample. The skilled artisan will understand that separation in such columns is a diffusional process. A variety of column packing materials are available for chromatographic separation of samples, and selection of an appropriate separation protocol is an empirical process that depends on the sample characteristics, the analyte of interest, the interfering substances present and their characteristics, etc. Various packing chemistries can be used depending on the needs (e.g., structure, polarity, and solubility of compounds being purified). In various embodiments the columns are polar, ion exchange (both cation and anion), hydrophobic interaction, phenyl, C-2, C-8, C-18 columns, polar coating on porous polymer, or others that are commercially available. During chromatography, the separation of materials is effected by variables such as choice of eluant (also known as a “mobile phase”), choice of gradient elution and the gradient conditions, temperature, etc. In certain embodiments, an analyte may be purified by applying a sample to a column under conditions where the analyte of interest is reversibly retained by the column packing material, while one or more other materials are not retained. In these embodiments, a first mobile phase condition can be employed where the analyte of interest is retained by the column, and a second mobile phase condition can subsequently be employed to remove retained material from the column, once the non-retained materials are washed through. Alternatively, an analyte may be purified by applying a sample to a column under mobile phase conditions where the analyte of interest elutes at a differential rate in comparison to one or more other materials. As discussed above, such procedures may enrich the amount of one or more analytes of interest relative to one or more other components of the sample.

The following parameters are used to specify an LC-SRM-MS assay of a protein under a particular LC-SRM-MS system: (1) a tryptic peptide of the protein; (2) the retention time (RT) of the peptide; (3) the m/z value of the peptide precursor ion; (4) the declustering potential used to ionize the precursor ion; (5) the m/z value of a fragment ion generated from the peptide precursor ion; and (6) the collision energy (CE) used to fragment the peptide precursor ion that is optimized for the particular peptide.

In certain embodiments of the preceding methods, the two best peptides per protein and the two best transitions per peptide are selected based on experimental data resulting from LC-SRM-MS analysis of one or more of the following experimental samples: a biological disease sample, a biological control sample, and a mixture of synthetic peptides of interest. Biological samples include body fluids, tissue samples and cell samples. Body fluid samples can include blood, serum, sputum, genital secretions, cerebrospinal fluid, sweat or excreta such as urine. Body tissue samples can include lung, skin, brain, spine, bone, muscle, epithelial, liver, kidney, pancreas, gastrointestinal tract, cardiovascular tissue, heart or nervous tissue. Biological disease samples can include cancer, benign tumors, infected tissue and tissue subject to trauma. In a particular embodiment, the biological disease and biological control samples are processed using an immunodepletion method prior to LC-SRM-MS analysis. Immunodepletion involves removal of one or more proteins through the use of antibodies. Numerous immunodepletion techniques are known to those of skill in the art. In another embodiment, the biological disease and biological control samples are processed using an immunocapture method prior to LC-SRM-MS analysis. Immunocapture involves selection of one or more proteins through the use of antibodies. Numerous immunocapture techniques are known to those of skill in the art.

To facilitate accurate quantification of the peptide transitions by the methods disclosed herein, a set of isotopically-labeled synthetic versions of the peptides of interest may be added in known amounts to the sample for use as internal standards. Since the isotopically-labeled peptides have physical and chemical properties identical to the corresponding surrogate peptide, they co-elute from the chromatographic column and are easily identifiable on the resultant mass spectrum. The addition of the labeled standards may occur before or after proteolytic digestion. Methods of synthesizing isotopically-labeled peptides will be known to those of skill in the art. Thus, in another embodiment, the experimental samples contain internal standard peptides. Other embodiments may utilize external standards or other expedients for peptide quantification.

In yet another embodiment, the LC-SRM-MS analysis method specifies a maximum of 7000 transitions, including transitions of the internal standard peptides and transitions. As used herein, the term “transition” refers to the specific pair of m/z (mass-to-charge) values associated with the precursor and transition ions corresponding to a specific peptide and, therefore, to a specific protein.

In one embodiment of the method, the top two transitions per peptide are selected according to one or more of the following criteria (A): (1) the transitions exhibit the largest peak areas measured in either of the two biological experimental samples; (2) the transitions are not interfered with by other ions; (3) the transitions do not exhibit an elution profile that visually differs from those of other transitions of the same peptide; (4) the transitions are not beyond the detection limit of both of the two biological experimental samples; (5) the transitions do not exhibit interferences.

For the mass spectrometric analysis of a particular peptide, the quantities of the peptide transitions in the sample may be determined by integration of the relevant mass spectral peak areas, as known in the prior art. When isotopically-labeled internal standards are used, as described above, the quantities of the peptide transitions of interest are established via an empirically-derived or predicted relationship between peptide transition quantity (which may be expressed as concentration) and the area ratio of the peptide transition and internal standard peaks at specified transitions.

In another embodiment of the method, the top two peptides per protein are selected according to one or more of the following criteria (B): (1) one or more peptides exhibit two transitions according to criteria (A) and represent the largest combined peak areas of the two transitions according to criteria (A); and (2) one or more peptides exhibit one transition according to criteria (A) and represent the largest combined peak areas of the two transitions according to criteria (A).

Assays

The methods of the present disclosure allow the quantification of high abundance and low abundance plasma proteins that serve as detectable markers for various health states (including diseases and disorders), thus forming the basis for assays that can be used to determine the differences between normal levels of detectable markers and changes of such detectable markers that are indicative of changes in health status. In one aspect of the invention, provided herein is an assay developed according to the foregoing method, and embodiments thereof. In another aspect, provided herein is the use of an assay developed according to the foregoing method, and embodiments thereof, to detect a plurality of at least 200, 300, or more proteins in a single sample. In a merely illustration embodiment, 388 proteins in the following table 1 are detected utilizing the method of present invention.

Of the 388 proteins, the 36 most cooperative proteins are listed in Table 2.

SRM assays for the 388 proteins were developed using standard synthetic peptide techniques. Of the 388 candidates, SRM assays were successfully developed for 371 candidates. The 371 SRM assays were applied to benign and lung cancer plasma samples to evaluate detection rate in blood. The summary of the SRM assay for these 371 proteins is listed in table 3 (see also Example III).

Definitions

As used herein, “transition” refers to a pair of m/z values associated with a peptide. Normally, labeled synthetic peptides are used as quality controls in SRM assays. However, for very large SRM assays such as the 371 protein lung cancer assay, labeled peptides are not feasible. However, correlation techniques (Kearney, Butler et al. 2008) were used to confirm the identity of protein transitions with high confidence. In FIG. 2 a histogram of the Pearson correlations between every pair of transitions in the assay is presented. The correlation between a pair of transitions is obtained from their expression profiles over all samples (143) in the training study detailed below. As expected, transitions from the same peptide are highly correlated. Similarly, transitions from different peptide fragments of the same protein are also highly correlated. In contrast, transitions from different proteins are not highly correlated. This methodology enables a statistical analysis of the quality of a protein's SRM assay. For example, if the correlation of transitions from two peptides from the same protein is above 0.5 then there is less than a 5% probability that the assay is false.

As used herein, a “tryptic peptide” refers to the peptide that is formed by the treatment of a protein with trypsin.

As used herein, “RT” refers to “retention time”, the elapsed time between injection and elution of an analyte.

As used herein, “m/z” indicates the mass-to-charge ratio of an ion.

As used herein, “DP” refers to “declustering potential”, a voltage potential to dissolvate and dissociate ion clusters. It is also known as “fragmentor voltage” or “ion transfer capillary offset voltage” depending on the manufacture.

As used herein, “CE” refers to “collision energy”, the amount of energy precursor ions receive as they are accelerated into the collision cell.

As used herein, “LC-SRM-MS” is an acronym for “selected reaction monitoring” and may be used interchangeably with “LC-MRM-MS”.

As used herein, “MS/MS” represents tandem mass spectrometry, which is a type of mass spectrometry involving multiple stages of mass analysis with some form of fragmentation occurring in between the stages.

As used herein, “ISP” refers to “internal standard peptides”.

As used herein, “HGS” refers to “human gold standard”, which is comprised of a pool of plasma from healthy individuals.

As used herein, “MGF” refers to “Mascot generic file”. Mascot is a search engine that uses mass spectrometry data to identify proteins from primary sequence databases. A Mascot generic file is a plain text (ASCII) file containing peak list information and, optionally, search parameters.

Mascot is a web-based tool for deriving protein sequences from mass spectrometry data. This data can be acquired from any mass spectrometry technique including MALDI-TOF and MS/MS (including LC-SRM-MS) data. Mascot uses a ‘probability-based MOWSE’ algorithm to estimate the significance of a match (i.e., that the observed transitions correspond to a particular protein). The total score is the absolute probability that the observed match is a random event. They are reported as −10×LOG10(P), where P is the absolute probability. Lower probabilities, therefore, are reported as higher scores. For example, if the absolute probability that an observed match is random is 1×10⁻¹², Mascot reports it as 120.

The disclosure also provides compositions. These compositions can include any of the transition ions described in Table 2. These transition ions exist while peptides derived from the proteins in Table 2 are undergoing analysis with LC-SRM-MS. In one embodiment, the composition includes any of the transition ions described in Table 2. In another embodiment, the composition includes any two transition ions described in Table 2. In other embodiments, the composition includes, any 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or 331 transition ions described in Table 2.

In another embodiment, the transition ions correspond with human proteins including LRP1, BGH3, COIA1, TETN, TSP1, ALDOA, GRP78, ISLR, FRIL, LG3BP, PRDX1, FIBA, and GSLG1. In another embodiment, the transition ions are derived from human proteins including LRP1, BGH3, COIA1, TETN, TSP1, ALDOA, GRP78, ISLR, FRIL, LG3BP, PRDX1, FIBA, and GSLG1. These proteins can further include transition ions corresponding with and/or derived from any number of additional proteins from Table 2. Thus, the composition can include, any additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or 331 transition ions described in Table 2.

In another embodiment, each of the transition ions in the composition corresponds and/or is derived from a different protein. In another embodiment, 90% of the transition ions in the composition correspond with and/or are derived from a protein that no other transition ion in the composition corresponds. In other embodiments, 80, 70, 60, 50, 40, 30, 20, 10 or 0% of the transition ions in the composition correspond and/or are derived from a protein that no other transition ion in the composition corresponds.

The compositions described herein included synthetic peptides. Synthetic peptides can be used as controls for the abundance of proteins they are derived from and/or correspond. In certain embodiments, the abundance of the synthetic peptides is defined and the results are compared to LC-SRM-MS results from a peptide found in a sample to the LC-SRM-MS results in the corresponding synthetic peptide. This allows for the calculation of the abundance of the peptide in the sample. In certain embodiments, by knowing the abundance of a peptide in a sample, the abundance of the protein it corresponded to is determined.

Synthetic peptides can be generated using any method known in the art. These methods can include recombinant expression techniques such as expression in bacteria or in vitro expression in eukaryotic cell lysate. These methods can also include solid phase synthesis.

In one embodiment, the composition includes synthetic peptides selected from any of the peptides described in Table 2. In another embodiment, the composition included any two peptides described in Table 2. In other embodiments, the composition included, any 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or more peptides described in Table 2.

In another embodiment, the peptides corresponded with human genes including LRP1, BGH3, COIA1, TETN, TSP1, ALDOA, GRP78, ISLR, FRIL, LG3BP, PRDX1, FIBA, and GSLG1 as described in corresponding patent application. These genes can further include peptides corresponding with any number of additional genes from Table 2. Thus, the composition can include, any additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or more peptides described in Table 2.

In another embodiment, each of the peptides in the composition each corresponds with a different protein. In another embodiment, 90% of the peptides in the composition correspond with a protein that no other peptide in the composition corresponds with. In other embodiments, 80, 70, 60, 50, 40, 30, 20, 10 or 0% of the peptides in the composition correspond with from a protein that no other peptide in the composition corresponds with.

The peptides can be isotopically labeled. The isotopes with which they can be labeled include ¹³C, ²H, ¹⁵N and ¹⁸O. The peptides can also include a polar solvent. Polar solvents can include water and mixtures of ethanol and water.

In certain embodiments, the samples described herein are taken from mammals. These mammals include rats, mice, rabbits, dogs, non-human primates and humans. Samples can be isolated from any tissue or organ or from any bodily fluid. Organs from which samples can be taken include skin, heart, lung, brain, kidney, liver, pancreas, spleen, testes, ovaries, gall bladder, thymus, thyroid, eye, ear, nose, mouth, tongue, penis, vagina, bladder or larynx. Tissues include nervous tissue, vascular tissue, muscle, bone, gastrointestinal tract, epithelial tissue, fibroblastic tissue, mucous membranes, hair, skin, reproductive tissue and connective tissue. Body fluids and excretions include, blood, serum, saliva, urine, semen, vaginal secretions, excrement, bile, tears, lymph, ear wax, mucous, shed skin, finger nails, toe nails, skin oils, sweat and dandruff.

The relative abundance of one or more of the proteins represented by the transition ions and synthetic peptides described above can be used to diagnose, determine likelihood of the presence of, develop prognoses for and/or stage various diseases and pathologies. Often the organ, tissue or bodily fluid or excretion from which the sample is taken is distinct from the organ, tissue or bodily fluid or excretion involved with the disease or pathology. For example, the presence of lung cancer can be determined from a sample taken from blood. Any type of body fluid may be used in the assays.

Diseases and pathologies that status, diagnosis, presence or prognosis can be found using the transition ions and/or synthetic peptides described herein include cancer, metabolic diseases, neurological disorders, infectious diseases and cardiovascular disorders.

EXAMPLES Exemplary Standard Operating Procedure

Protein Selection

Proteins known to be over-expressed on the cell surface of lung cancer tumors were obtained (through literature searching, experimental data or proprietary databases). This was referred to as set ‘A’.

Proteins known to be over-secreted by lung cancer tumor cells were obtained (through literature searching, experimental data or proprietary databases). This was referred to as set ‘B’. Proteins associated with lung cancer in the literature were mined. This was referred to as set ‘C’. Proteins of interest (sets A, B and C are merged resulting in over 700 proteins) were assembled. The set of proteins was reduced to a set of 388 proteins (see Table 4) by prioritizing those proteins that have been previously detected my LC-MS/MS in blood (serum or plasma).

Selected proteins were then identified by their UniProt protein name and accession, their Entrez gene symbol and gene name, the isoform accession and their amino acid sequence. The canonical isoform in UniProt was selected if a protein has more than one isoform.

Peptide Selection for Synthesis

The five best peptides per protein for LC-SRM-MS assay were selected for as follows. Fully tryptic peptides having a monoisotopic mass of 800-3500 mass units, without miscleavages, not containing a cysteine (C) or a methionine (M), without having high miscleavage probability were selected. Further, any peptide that was bounded by KK, KR, RK or RR (either upstream or downstream) in the corresponding protein sequence was not selected.

Peptides were selected that were unique to the protein of interest. Peptides were only selected that match only one protein or protein family including analogues of the one protein, when searched in protein databases. Further, peptides which were observed in post-translational modified forms were not selected. Databases were assessed that showed expression of the proteins from which the peptides were isolated in human blood. Also databases of good quality MS peptides were searched. Peptides that appeared in human blood and were good quality MS peptides were favored. If these methods did not result in a sufficient number of peptides, rules were relaxed in a step wise manner to allow a greater number of peptides until a sufficient number was reached. The purity of the synthesized peptides was >75% and the amount of material was ≥25 μg. Peptides did not need to be desalted.

The four best transitions per peptide are then selected and optimized based on experimental results from a mixture of synthetic peptides. LC-SRM-MS-triggered MS/MS spectra were acquired for each synthetic peptide, using a QTRAP 5500 instrument. One spectrum for the doubly- and one for the triply-charged precursor ion was collected for each peptide For the identified peptides (Mascot score ≥15), retention time was recorded for the four most intense b or y transition ions. The selected transition ions possessed m/z values were at least 30 m/z above or below those of the precursor ions; they did not interfere with other synthetic peptides; and they were transition ions due to breakage of peptide bond at different sites.

If an insufficient percentage of the synthetic peptides were acquired, the steps were repeated. In some cases, the second transition with first with theoretical y+ ions with m/z values at least 30 m/z above those of the doubly charged precursor ion was selected if an insufficient percentage was acquired. Peptides that failed to trigger the acquisition of MS/MS spectrum were discarded.

Collision energy (CE) for each selected transition (See Table 4) was optimized.

Exemplary Protein List

The abundance of the following proteins can be assessed substantially simultaneously using the MS-LC-SRM-MS system described herein. Transitions from these proteins can be used to diagnose diseases including lung cancer when their abundance is measured in a biological specimen from a subject to be diagnosed for lung cancer. In one embodiment, the abundances of these proteins are measured in the blood serum of the subject.

TABLE 1 Sources of Subcellular Evidence for Gene Tissue Biomarkers Location Presence in UniProt Protein Protein Name Symbol Biomarkers in Literature (UniProt) Blood 1433B_HUMAN 14-3-3 YWHAB Secreted, LungCancers Cytoplasm. Literature, protein EPI Melanosome. Detection beta/alpha Note = Identified by mass spectrometry in melanosome fractions from stage I to stage IV. 1433E_HUMAN 14-3-3 YWHAE ENDO LungCancers, Cytoplasm (By Literature, protein BenignNodules similarity). Detection epsilon Melanosome. Note = Identified by mass spectrometry in melanosome fractions from stage I to stage IV. 1433S_HUMAN 14-3-3 SFN Secreted, LungCancers Cytoplasm. UniProt, protein EPI Nucleus (By Literature, sigma similarity). Detection Secreted. Note = May be secreted by a non-classical secretory pathway. 1433T_HUMAN 14-3-3 YWHAQ EPI LungCancers, Cytoplasm. Detection protein BenignNodules Note = In theta neurons, axonally transported to the nerve terminals. 1433Z_HUMAN 14-3-3 YWHAZ EPI LungCancers, Cytoplasm. Detection protein BenignNodules Melanosome. zeta/delta Note = Located to stage I to stage IV melanosomes. 6PGD_HUMAN 6- PGD EPI, ENDO Cytoplasm (By Detection phosphogluconate similarity). dehydrogenase, decarboxylating A1AG1_HUMAN Alpha-1- ORM1 EPI Symptoms Secreted. UniProt, acid Literature, glycoprotein 1 Detection, ABCD1_HUMAN ATP- ABCD1 ENDO Peroxisome Detection, binding membrane; Prediction cassette Multi-pass sub-family membrane D member protein. 1 ADA12_HUMAN Disintegrin ADAM12 LungCancers, Isoform 1: Cell UniProt, and BenignNodules, membrane; Detection, metalloproteinase Symptoms Single-pass Prediction domain- type I containing membrane protein 12 protein.|Isoform 2: Secreted.|Isoform 3: Secreted (Potential).|Isoform 4: Secreted (Potential). ADML_HUMAN ADM ADM LungCancers, Secreted. UniProt, BenignNodules, Literature, Symptoms Detection, Prediction AGR2_HUMAN Anterior AGR2 EPI LungCancers Secreted. UniProt, gradient Endoplasmic Prediction protein 2 reticulum (By homolog similarity). AIFM1_HUMAN Apoptosis- AIFM1 EPI, ENDO LungCancers Mitochondrion Detection, inducing intermembrane Prediction factor 1, space. Nucleus. mitochondrial Note = Translocated to the nucleus upon induction of apoptosis. ALDOA_HUMAN Fructose- ALDOA Secreted, LungCancers, Literature, bisphosphate EPI Symptoms Detection aldolase A AMPN_HUMAN Aminopeptidase ANPEP EPI, ENDO LungCancers, Cell membrane; UniProt, N BenignNodules, Single-pass Detection Symptoms type II membrane protein. Cytoplasm, cytosol (Potential). Note = A soluble form has also been detected. ANGP1_HUMAN Angiopoietin- ANGPT1 LungCancers, Secreted. UniProt, 1 BenignNodules Literature, Prediction ANGP2_HUMAN Angiopoietin- ANGPT2 LungCancers, Secreted. UniProt, 2 BenignNodules Literature, Prediction APOA1_HUMAN Apolipoprotein APOA1 LungCancers, Secreted. UniProt, A-I BenignNodules, Literature, Symptoms Detection, Prediction APOE_HUMAN Apolipoprotein APOE EPI, ENDO LungCancers, Secreted. UniProt, E BenignNodules, Literature, Symptoms Detection, Prediction ASM3B_HUMAN Acid SMPDL3B EPI, ENDO Secreted (By UniProt, sphingomyelinase- similarity). Prediction like phosphodiesterase 3b AT2A2_HUMAN Sarcoplasmic/ ATP2A2 EPI, ENDO LungCancers, Endoplasmic Detection endoplasmic BenignNodules reticulum reticulum membrane; calcium Multi-pass ATPase 2 membrane protein. Sarcoplasmic reticulum membrane; Multi-pass membrane protein. ATS1_HUMAN A ADAMTS1 LungCancers, Secreted, UniProt, disintegrin BenignNodules, extracellular Literature, and Symptoms space, Prediction metalloproteinase extracellular with matrix (By thrombospondin similarity). motifs 1 ATS12_HUMAN A ADAMTS12 LungCancers Secreted, UniProt, disintegrin extracellular Detection, and space, Prediction metalloproteinase extracellular with matrix (By thrombospondin similarity). motifs 12 ATS19_HUMAN A ADAMTS19 LungCancers Secreted, UniProt, disintegrin extracellular Prediction and space, metalloproteinase extracellular with matrix (By thrombospondin similarity). motifs 19 BAGE1_HUMAN B BAGE LungCancers Secreted UniProt, melanoma (Potential). Prediction antigen 1 BAGE2_HUMAN B BAGE2 LungCancers Secreted UniProt, melanoma (Potential). Prediction antigen 2 BAGE3_HUMAN B BAGE3 LungCancers Secreted UniProt, melanoma (Potential). Prediction antigen 3 BAGE4_HUMAN B BAGE4 LungCancers Secreted UniProt, melanoma (Potential). Prediction antigen 4 BAGE5_HUMAN B BAGE5 LungCancers Secreted UniProt, melanoma (Potential). Prediction antigen 5 BASP1_HUMAN Brain acid BASP1 Secreted, Cell membrane; Detection soluble EPI Lipid-anchor. protein 1 Cell projection, growth cone. Note = Associated with the membranes of growth cones that form the tips of elongating axons. BAX_HUMAN Apoptosis BAX EPI LungCancers, Isoform Alpha: UniProt, regulator BenignNodules Mitochondrion Literature, BAX membrane; Prediction Single-pass membrane protein. Cytoplasm. Note = Colocalizes with 14- 3-3 proteins in the cytoplasm. Under stress conditions, redistributes to the mitochondrion membrane through the release from JNK- phosphorylated 14-3-3 proteins.|Isoform Beta: Cytoplasm.|Isoform Gamma: Cytoplasm.|Isoform Delta: Cytoplasm (Potential). BDNF_HUMAN Brain- BDNF BenignNodules, Secreted. UniProt, derived Symptoms Literature, neurotrophic Prediction factor BGH3_HUMAN Transforming TGFBI LungCancers, Secreted, UniProt, growth BenignNodules extracellular Detection factor-beta- space, induced extracellular protein ig- matrix. h3 Note = May be associated both with microfibrils and with the cell surface. BMP2_HUMAN Bone BMP2 LungCancers, Secreted. UniProt, morphogenetic BenignNodules, Literature protein Symptoms 2 BST1_HUMAN ADP- BST1 EPI Symptoms Cell membrane; Detection, ribosyl Lipid-anchor, Prediction cyclase 2 GPI-anchor. C163A_HUMAN Scavenger CD163 EPI Symptoms Soluble CD163: UniProt, receptor Secreted.|Cell Detection cysteine- membrane; rich type 1 Single-pass protein type I M130 membrane protein. Note = Isoform 1 and isoform 2 show a lower surface expression when expressed in cells. C4BPA_HUMAN C4b- C4BPA LungCancers, Secreted. UniProt, binding Symptoms Detection, protein Prediction alpha chain CAH9_HUMAN Carbonic CA9 LungCancers, Nucleus. UniProt anhydrase BenignNodules, Nucleus, 9 Symptoms nucleolus. Cell membrane; Single-pass type I membrane protein. Cell projection, microvillus membrane; Single-pass type I membrane protein. Note = Found on the surface microvilli and in the nucleus, particularly in nucleolus. CALR_HUMAN Calreticulin CALR EPI Symptoms Endoplasmic UniProt, reticulum Literature, lumen. Detection, Cytoplasm, Prediction cytosol. Secreted, extracellular space, extracellular matrix. Cell surface. Note = Also found in cell surface (T cells), cytosol and extracellular matrix. Associated with the lytic granules in the cytolytic T- lymphocytes. CALU_HUMAN Calumenin CALU EPI Symptoms Endoplasmic UniProt, reticulum Detection, lumen. Prediction Secreted. Melanosome. Sarcoplasmic reticulum lumen (By similarity). Note = Identified by mass spectrometry in melanosome fractions from stage I to stage IV. CALX_HUMAN Calnexin CANX Secreted, BenignNodules Endoplasmic UniProt, EPI, ENDO reticulum Literature, membrane; Detection Single-pass type I membrane protein. Melanosome. Note = Identified by mass spectrometry in melanosome fractions from stage I to stage IV. CAP7_HUMAN Azurocidin AZU1 EPI Symptoms Cytoplasmic Prediction granule. Note = Cytoplasmic granules of neutrophils. CATB_HUMAN Cathepsin CTSB Secreted LungCancers Lysosome. Literature, B Melanosome. Detection, Note = Identified Prediction by mass spectrometry in melanosome fractions from stage I to stage IV. CATG_HUMAN Cathepsin CTSG Secreted, BenignNodules Cell surface. Detection, G ENDO Prediction CBPB2_HUMAN Carboxypeptidase CPB2 LungCancers, Secreted. UniProt, B2 BenignNodules, Detection, Symptoms Prediction CCL22_HUMAN C-C motif CCL22 LungCancers, Secreted. UniProt, chemokine 22 BenignNodules Prediction CD14_HUMAN Monocyte CD14 EPI LungCancers, Cell membrane; Literature, differentiation BenignNodules, Lipid-anchor, Detection, antigen Symptoms GPI-anchor. Prediction CD14 CD24_HUMAN Signal CD24 LungCancers, Cell membrane; Literature transducer BenignNodules Lipid-anchor, CD24 GPI-anchor. CD2A2_HUMAN Cyclin- CDKN2A LungCancers, Cytoplasm. Literature, dependent BenignNodules Nucleus.|Nucleus, Prediction kinase nucleolus inhibitor 2A, (By similarity). isoform 4 CD38_HUMAN ADP- CD38 EPI, ENDO Symptoms Membrane; UniProt, ribosyl Single-pass Literature cyclase 1 type II membrane protein. CD40L_HUMAN CD40 CD40LG LungCancers, Cell membrane; UniProt, ligand BenignNodules, Single-pass Literature Symptoms type II membrane protein.|CD40 ligand, soluble form: Secreted. CD44_HUMAN CD44 CD44 EPI LungCancers, Membrane; UniProt, antigen BenignNodules, Single-pass Literature, Symptoms type I Detection, membrane Prediction protein. CD59_HUMAN CD59 CD59 LungCancers, Cell membrane; UniProt, glycoprotein BenignNodules, Lipid-anchor, Literature, Symptoms GPI-anchor. Detection, Secreted. Prediction Note = Soluble form found in a number of tissues. CD97_HUMAN CD97 CD97 EPI, ENDO Symptoms Cell membrane; UniProt antigen Multi-pass membrane protein.|CD97 antigen subunit alpha: Secreted, extracellular space. CDCP1_HUMAN CUB CDCP1 LungCancers Isoform 1: Cell UniProt, domain- membrane; Prediction containing Single-pass protein 1 membrane protein (Potential). Note = Shedding may also lead to a soluble peptide.|Isoform 3: Secreted. CDK4_HUMAN Cell CDK4 LungCancers, Literature division Symptoms protein kinase 4 CEAM5_HUMAN Carcinoembryonic CEACAM5 EPI LungCancers, Cell membrane; Literature, antigen- BenignNodules, Lipid-anchor, Prediction related cell Symptoms GPI-anchor. adhesion molecule 5 CEAM8_HUMAN Carcinoembryonic CEACAM8 EPI LungCancers Cell membrane; Detection, antigen- Lipid-anchor, Prediction related cell GPI-anchor. adhesion molecule 8 CERU_HUMAN Ceruloplasmin CP EPI LungCancers, Secreted. UniProt, Symptoms Literature, Detection, Prediction CH10_HUMAN 10 kDa HSPE1 ENDO LungCancers Mitochondrion Literature, heat shock matrix. Detection, protein, Prediction mitochondrial CH60_HUMAN 60 kDa HSPD1 Secreted, LungCancers, Mitochondrion Literature, heat shock EPI, ENDO Symptoms matrix. Detection protein, mitochondrial CKAP4_HUMAN Cytoskeleton- CKAP4 EPI, ENDO LungCancers Endoplasmic UniProt associated reticulum-Golgi protein 4 intermediate compartment membrane; Single-pass membrane protein (Potential). CL041_HUMAN Uncharacterized C12orf41 ENDO Prediction protein C12orf41 CLCA1_HUMAN Calcium- CLCA1 LungCancers, Secreted, UniProt, activated BenignNodules extracellular Prediction chloride space. Cell channel membrane; regulator 1 Peripheral membrane protein; Extracellular side. Note = Protein that remains attached to the plasma membrane appeared to be predominantly localized to microvilli. CLIC1_HUMAN Chloride CLIC1 EPI Nucleus. UniProt, intracellular Nucleus Literature, channel membrane; Detection protein 1 Single-pass membrane protein (Probable). Cytoplasm. Cell membrane; Single-pass membrane protein (Probable). Note = Mostly in the nucleus including in the nuclear membrane. Small amount in the cytoplasm and the plasma membrane. Exists both as soluble cytoplasmic protein and as membrane protein with probably a single transmembrane domain. CLUS_HUMAN Clusterin CLU EPI, ENDO LungCancers, Secreted. UniProt, BenignNodules, Literature, Symptoms Detection, Prediction CMGA_HUMAN Chromogranin- CHGA LungCancers, Secreted. UniProt, A BenignNodules Note = Literature, Neuroendocrine and Detection, endocrine Prediction secretory granules. CNTN1_HUMAN Contactin-1 CNTN1 LungCancers Isoform 1: Cell Detection, membrane; Prediction Lipid-anchor, GPI- anchor; Extracellular side.|Isoform 2: Cell membrane; Lipid-anchor, GPI- anchor; Extracellular side. CO4A1_HUMAN Collagen COL4A1 LungCancers Secreted, UniProt, alpha-1(IV) extracellular Detection, chain space, Prediction extracellular matrix, basement membrane. CO5A2_HUMAN Collagen COL5A2 LungCancers Secreted, UniProt, alpha-2(V) extracellular Detection, chain space, Prediction extracellular matrix (By similarity). CO6A3_HUMAN Collagen COL6A3 Secreted Symptoms Secreted, UniProt, alpha-3(VI) extracellular Detection, chain space, Prediction extracellular matrix (By similarity). COCA1_HUMAN Collagen COL12A1 ENDO LungCancers, Secreted, UniProt, alpha- Symptoms extracellular Prediction 1(XII) space, chain extracellular matrix (By similarity). COF1_HUMAN Cofilin-1 CFL1 Secreted, LungCancers, Nucleus matrix. Detection, EPI BenignNodules Cytoplasm, Prediction cytoskeleton. Note = Almost completely in nucleus in cells exposed to heat shock or 10% dimethyl sulfoxide. COIA1_HUMAN Collagen COL18A1 LungCancers, Secreted, UniProt, alpha- BenignNodules extracellular Literature, 1(XVIII) space, Detection, chain extracellular Prediction matrix (By similarity). COX5A_HUMAN Cytochrome COX5A Secreted, Mitochondrion Prediction c oxidase ENDO inner subunit 5A, membrane. mitochondrial CRP_HUMAN C-reactive CRP LungCancers, Secreted. UniProt, protein BenignNodules, Literature, Symptoms Detection, Prediction CS051_HUMAN UPF0470 C19orf51 ENDO Prediction protein C19orf51 CSF1_HUMAN Macrophage CSF1 LungCancers, Cell membrane; UniProt, colony- BenignNodules Single-pass Literature, stimulating membrane Detection factor 1 protein (By similarity).|Processed macrophage colony- stimulating factor 1: Secreted, extracellular space (By similarity). CSF2_HUMAN Granulocyte- CSF2 LungCancers, Secreted. UniProt, macrophage BenignNodules Literature, colony- Prediction stimulating factor CT085_HUMAN Uncharacterized C20orf85 LungCancers, Prediction protein BenignNodules C20orf85 CTGF_HUMAN Connective CTGF LungCancers, Secreted, UniProt, tissue BenignNodules extracellular Literature, growth space, Detection, factor extracellular Prediction matrix (By similarity). Secreted (By similarity). CYR61_HUMAN Protein CYR61 LungCancers, Secreted. UniProt, CYR61 BenignNodules Prediction CYTA_HUMAN Cystatin-A CSTA LungCancers Cytoplasm. Literature, Detection CYTB_HUMAN Cystatin-B CSTB Secreted Cytoplasm. Literature, Nucleus. Detection DDX17_HUMAN Probable DDX17 ENDO LungCancers, Nucleus. Detection, ATP- BenignNodules Prediction dependent RNA helicase DDX17 DEFB1_HUMAN Beta- DEFB1 LungCancers, Secreted. UniProt, defensin 1 BenignNodules Prediction DESP_HUMAN Desmoplakin DSP EPI, ENDO LungCancers Cell junction, Detection desmosome. Cytoplasm, cytoskeleton. Note = Innermost portion of the desmosomal plaque. DFB4A_HUMAN Beta- DEFB4A LungCancers, Secreted. UniProt defensin BenignNodules 4A DHI1L_HUMAN Hydroxysteroid HSD11B1L LungCancers Secreted UniProt, 11- (Potential). Prediction beta- dehydrogenase 1-like protein DMBT1_HUMAN Deleted in DMBT1 LungCancers, Secreted (By UniProt, malignant BenignNodules similarity). Detection, brain Note = Some Prediction tumors 1 isoforms may protein be membrane- bound. Localized to the lumenal aspect of crypt cells in the small intestine. In the colon, seen in the lumenal aspect of surface epithelial cells. Formed in the ducts of von Ebner gland, and released into the fluid bathing the taste buds contained in the taste papillae (By similarity). DMKN_HUMAN Dermokine DMKN LungCancers Secreted. UniProt, Detection, Prediction DPP4_HUMAN Dipeptidyl DPP4 EPI LungCancers, Dipeptidyl UniProt, peptidase 4 BenignNodules, peptidase 4 Detection Symptoms soluble form: Secreted.|Cell membrane; Single-pass type II membrane protein. DSG2_HUMAN Desmoglein- DSG2 ENDO Symptoms Cell membrane; UniProt, 2 Single-pass Detection type I membrane protein. Cell junction, desmosome. DX39A_HUMAN ATP- DDX39A EPI Nucleus (By Prediction dependent similarity). RNA helicase DDX39A DX39B_HUMAN Spliceosome DDX39B EPI Nucleus. Prediction RNA Nucleus helicase speckle. DDX39B DYRK2_HUMAN Dual DYRK2 ENDO LungCancers Cytoplasm. Literature specificity Nucleus. tyrosine- Note = Translocates phosphorylation- into the regulated nucleus kinase 2 following DNA damage. EDN2_HUMAN Endothelin-2 EDN2 LungCancers Secreted. UniProt, Prediction EF1A1_HUMAN Elongation EEF1A1 Secreted, LungCancers, Cytoplasm. Detection factor 1- EPI BenignNodules alpha 1 EF1D_HUMAN Elongation EEF1D Secreted, LungCancers Prediction factor 1- EPI delta EF2_HUMAN Elongation EEF2 Secreted, Cytoplasm. Literature, factor 2 EPI Detection EGF_HUMAN Pro- EGF LungCancers, Membrane; UniProt, epidermal BenignNodules, Single-pass Literature growth Symptoms type I factor membrane protein. EGFL6_HUMAN Epidermal EGFL6 LungCancers Secreted, UniProt, growth extracellular Detection, factor-like space, Prediction protein 6 extracellular matrix, basement membrane (By similarity). ENOA_HUMAN Alpha- ENO1 Secreted, LungCancers, Cytoplasm. Cell Literature, enolase EPI, ENDO BenignNodules, membrane. Detection, Symptoms Cytoplasm, Prediction myofibril, sarcomere, M- band. Note = Can translocate to the plasma membrane in either the homodimeric (alpha/alpha) or heterodimeric (alpha/gamma) form. ENO1 is localized to the M- band.|Isoform MBP-1: Nucleus. ENOG_HUMAN Gamma- ENO2 EPI LungCancers, Cytoplasm (By Literature, enolase Symptoms similarity). Cell Detection, membrane (By Prediction similarity). Note = Can translocate to the plasma membrane in either the homodimeric (alpha/alpha) or heterodimeric (alpha/gamma) form (By similarity). ENOX2_HUMAN Ecto-NOX ENOX2 LungCancers Cell membrane. UniProt, disulfide- Secreted, Detection thiol extracellular exchanger space. 2 Note = Extracellular and plasma membrane- associated. ENPL_HUMAN Endoplasmin HSP90B1 Secreted, LungCancers, Endoplasmic Literature, EPI, ENDO BenignNodules, reticulum Detection, Symptoms lumen. Prediction Melanosome. Note = Identified by mass spectrometry in melanosome fractions from stage I to stage IV. EPHB6_HUMAN Ephrin EPHB6 LungCancers Membrane; UniProt, type-B Single-pass Literature receptor 6 type I membrane protein.|Isoform 3: Secreted (Probable). EPOR_HUMAN Erythropoietin EPOR LungCancers, Cell membrane; UniProt, receptor BenignNodules, Single-pass Literature, Symptoms type I Detection membrane protein.|Isoform EPOR-S: Secreted. Note = Secreted and located to the cell surface. ERBB3_HUMAN Receptor ERBB3 LungCancers, Isoform 1: Cell UniProt, tyrosine- BenignNodules membrane; Literature, protein Single-pass Prediction kinase type I erbB-3 membrane protein.|Isoform 2: Secreted. EREG_HUMAN Proepiregulin EREG LungCancers Epiregulin: UniProt Secreted, extracellular space.|Proepiregulin: Cell membrane; Single-pass type I membrane protein. ERO1A_HUMAN ERO1-like ERO1L Secreted, Symptoms Endoplasmic Prediction protein EPI, ENDO reticulum alpha membrane; Peripheral membrane protein; Lumenal side. Note = The association with ERP44 is essential for its retention in the endoplasmic reticulum. ESM1_HUMAN Endothelial ESM1 LungCancers, Secreted. UniProt, cell- BenignNodules Prediction specific molecule 1 EZRI_HUMAN Ezrin EZR Secreted LungCancers, Apical cell Literature, BenignNodules membrane; Detection, Peripheral Prediction membrane protein; Cytoplasmic side. Cell projection. Cell projection, microvillus membrane; Peripheral membrane protein; Cytoplasmic side. Cell projection, ruffle membrane; Peripheral membrane protein; Cytoplasmic side. Cytoplasm, cell cortex. Cytoplasm, cytoskeleton. Note = Localization to the apical membrane of parietal cells depends on the interaction with MPP5. Localizes to cell extensions and peripheral processes of astrocytes (By similarity). Microvillar peripheral membrane protein (cytoplasmic side). F10A1_HUMAN Hsc70- ST13 EPI Cytoplasm (By Detection, interacting similarity).|Cytoplasm Prediction protein (Probable). FAM3C_HUMAN Protein FAM3C EPI, ENDO Secreted UniProt, FAM3C (Potential). Detection FAS_HUMAN Fatty acid FASN EPI LungCancers, Cytoplasm. Literature, synthase BenignNodules, Melanosome. Detection Symptoms Note = Identified by mass spectrometry in melanosome fractions from stage I to stage IV. FCGR1_HUMAN High FCGR1A EPI LungCancers, Cell membrane; UniProt affinity BenignNodules, Single-pass immunoglobulin Symptoms type I gamma Fc membrane receptor I protein. Note = Stabilized at the cell membrane through interaction with FCER1G. FGF10_HUMAN Fibroblast FGF10 LungCancers Secreted UniProt, growth (Potential). Prediction factor 10 FGF2_HUMAN Heparin- FGF2 , LungCancers, Literature binding BenignNodules, growth Symptoms factor 2 FGF7_HUMAN Keratinocyte FGF7 LungCancers, Secreted. UniProt, growth BenignNodules Literature, factor Prediction FGF9_HUMAN Glia- FGF9 LungCancers Secreted. UniProt, activating Literature, factor Prediction FGFR2_HUMAN Fibroblast FGFR2 LungCancers, Cell membrane; UniProt, growth BenignNodules Single-pass Literature, factor type I Prediction receptor 2 membrane protein.|Isoform 14: Secreted.|Isoform 19: Secreted. FGFR3_HUMAN Fibroblast FGFR3 LungCancers Membrane; UniProt, growth Single-pass Literature, factor type I Prediction receptor 3 membrane protein. FGL2_HUMAN Fibroleukin FGL2 BenignNodules, Secreted. UniProt, Symptoms Detection, Prediction FHIT_HUMAN Bis(5′- FHIT LungCancers, Cytoplasm. Literature adenosyl)- BenignNodules, triphosphatase Symptoms FIBA_HUMAN Fibrinogen FGA LungCancers, Secreted. UniProt, alpha chain BenignNodules, Literature, Symptoms Detection, Prediction FINC_HUMAN Fibronectin FN1 Secreted, LungCancers, Secreted, UniProt, EPI, ENDO BenignNodules, extracellular Literature, Symptoms space, Detection, extracellular Prediction matrix. FKB11_HUMAN Peptidyl- FKBP11 EPI, ENDO Membrane; UniProt, prolyl cis- Single-pass Prediction trans membrane isomerase protein FKBP11 (Potential). FOLH1_HUMAN Glutamate FOLH1 ENDO LungCancers, Cell membrane; UniProt, carboxypeptidase Symptoms Single-pass Literature 2 type II membrane protein.|Isoform PSMA′: Cytoplasm. FOLR1_HUMAN Folate FOLR1 LungCancers Cell membrane; UniProt receptor Lipid-anchor, alpha GPI-anchor. Secreted (Probable). FOXA2_HUMAN Hepatocyte FOXA2 LungCancers Nucleus. Detection, nuclear Prediction factor 3- beta FP100_HUMAN Fanconi C17orf70 ENDO Symptoms Nucleus. Prediction anemia- associated protein of 100 kDa FRIH_HUMAN Ferritin FTH1 EPI LungCancers, Literature, heavy BenignNodules Detection, chain Prediction FRIL_HUMAN Ferritin FTL Secreted, BenignNodules, Literature, light chain EPI, ENDO Symptoms Detection G3P_HUMAN Glyceraldehyde- GAPDH Secreted, LungCancers, Cytoplasm. Detection 3- EPI, ENDO BenignNodules, Cytoplasm, phosphate Symptoms perinuclear dehydrogenase region. Membrane. Note = Postnuclear and Perinuclear regions. G6PD_HUMAN Glucose-6- G6PD Secreted, LungCancers, Literature, phosphate EPI Symptoms Detection 1- dehydrogenase G6PI_HUMAN Glucose-6- GPI Secreted, Symptoms Cytoplasm. UniProt, phosphate EPI Secreted. Literature, isomerase Detection GA2L1_HUMAN GAS2-like GAS2L1 ENDO Cytoplasm, Prediction protein 1 cytoskeleton (Probable). GALT2_HUMAN Polypeptide GALNT2 EPI, ENDO Golgi UniProt, N- apparatus, Detection acetylgalactosaminyl- Golgi stack transferase 2 membrane; Single-pass type II membrane protein. Secreted. Note = Resides preferentially in the trans and medial parts of the Golgi stack. A secreted form also exists. GAS6_HUMAN Growth GAS6 LungCancers Secreted. UniProt, arrest- Detection, specific Prediction protein 6 GDIR2_HUMAN Rho GDP- ARHGDIB EPI Cytoplasm. Detection dissociation inhibitor 2 GELS_HUMAN Gelsolin GSN LungCancers, Isoform 2: UniProt, BenignNodules Cytoplasm, Literature, cytoskeleton.|Isoform Detection, 1: Prediction Secreted. GGH_HUMAN Gamma- GGH LungCancers Secreted, UniProt, glutamyl extracellular Detection, hydrolase space. Prediction Lysosome. Melanosome. Note = While its intracellular location is primarily the lysosome, most of the enzyme activity is secreted. Identified by mass spectrometry in melanosome fractions from stage I to stage IV. GPC3_HUMAN Glypican-3 GPC3 LungCancers, Cell membrane; UniProt, Symptoms Lipid-anchor, Literature, GPI-anchor; Prediction Extracellular side (By similarity).|Secreted glypican-3: Secreted, extracellular space (By similarity). GRAN_HUMAN Grancalcin GCA EPI Cytoplasm. Prediction Cytoplasmic granule membrane; Peripheral membrane protein; Cytoplasmic side. Note = Primarily cytosolic in the absence of calcium or magnesium ions. Relocates to granules and other membranes in response to elevated calcium and magnesium levels. GREB1_HUMAN Protein GREB1 ENDO Membrane; UniProt, GREB1 Single-pass Prediction membrane protein (Potential). GREM1_HUMAN Gremlin-1 GREM1 LungCancers, Secreted UniProt, BenignNodules (Probable). Prediction GRP_HUMAN Gastrin- GRP LungCancers, Secreted. UniProt, releasing Symptoms Prediction peptide GRP78_HUMAN 78 kDa HSPA5 Secreted, LungCancers, Endoplasmic Detection, glucose- EPI, ENDO BenignNodules reticulum Prediction regulated lumen. protein Melanosome. Note = Identified by mass spectrometry in melanosome fractions from stage I to stage IV. GSLG1_HUMAN Golgi GLG1 EPI, ENDO BenignNodules Golgi apparatus UniProt apparatus membrane; protein 1 Single-pass type I membrane protein. GSTP1_HUMAN Glutathione GSTP1 Secreted LungCancers, Literature, S- BenignNodules, Detection, transferase Symptoms Prediction P GTR1_HUMAN Solute SLC2A1 EPI, ENDO LungCancers, Cell membrane; Literature carrier BenignNodules, Multi-pass family 2, Symptoms membrane facilitated protein (By glucose similarity). transporter Melanosome. member 1 Note = Localizes primarily at the cell surface (By similarity). Identified by mass spectrometry in melanosome fractions from stage I to stage IV. GTR3_HUMAN Solute SLC2A3 EPI Membrane; Detection carrier Multi-pass family 2, membrane facilitated protein. glucose transporter member 3 H2A1_HUMAN Histone HIST1H2AG Secreted Nucleus. Detection, H2A type 1 Prediction H2A1B_HUMAN Histone HIST1H2AB Secreted Nucleus. Detection, H2A type Prediction 1-B/E H2A1C_HUMAN Histone HIST1H2AC Secreted Nucleus. Literature, H2A type Detection, 1-C Prediction H2A1D_HUMAN Histone HIST1H2AD Secreted Nucleus. Detection, H2A type Prediction 1-D HG2A_HUMAN HLA class CD74 LungCancers, Membrane; UniProt, II BenignNodules, Single-pass Literature histocompatibility Symptoms type II antigen membrane gamma chain protein (Potential). HGF_HUMAN Hepatocyte HGF LungCancers, Literature, growth BenignNodules, Prediction factor Symptoms HMGA1_HUMAN High HMGA1 LungCancers, Nucleus. Literature mobility BenignNodules, group Symptoms protein HMG- I/HMG-Y HPRT_HUMAN Hypoxanthine- HPRT1 EPI Cytoplasm. Detection, guanine Prediction phosphoribosyl- transferase HPSE_HUMAN Heparanase HPSE LungCancers, Lysosome UniProt, BenignNodules, membrane; Prediction Symptoms Peripheral membrane protein. Secreted. Note = Secreted, internalised and transferred to late endosomes/lysosomes as a proheparanase. In lysosomes, it is processed into the active form, the heparanase. The uptake or internalisation of proheparanase is mediated by HSPGs. Heparin appears to be a competitor and retain proheparanase in the extracellular medium. HPT_HUMAN Haptoglobin HP LungCancers, Secreted. UniProt, BenignNodules, Literature, Symptoms Detection, Prediction HS90A_HUMAN Heat shock HSP90AA1 Secreted, LungCancers, Cytoplasm. Literature, protein EPI Symptoms Melanosome. Detection HSP 90- Note = Identified alpha by mass spectrometry in melanosome fractions from stage I to stage IV. HS90B_HUMAN Heat shock HSP90AB1 Secreted, LungCancers Cytoplasm. Literature, protein EPI Melanosome. Detection HSP 90- Note = Identified beta by mass spectrometry in melanosome fractions from stage I to stage IV. HSPB1_HUMAN Heat shock HSPB1 Secreted, LungCancers, Cytoplasm. Literature, protein EPI BenignNodules Nucleus. Detection, beta-1 Cytoplasm, Prediction cytoskeleton, spindle. Note = Cytoplasmic in interphase cells. Colocalizes with mitotic spindles in mitotic cells. Translocates to the nucleus during heat shock. HTRA1_HUMAN Serine HTRA1 LungCancers Secreted. UniProt, protease Prediction HTRA1 HXK1_HUMAN Hexokinase- HK1 ENDO Symptoms Mitochondrion Literature, 1 outer Detection membrane. Note = Its hydrophobic N- terminal sequence may be involved in membrane binding. HYAL2_HUMAN Hyaluronidase- HYAL2 LungCancers Cell membrane; Prediction 2 Lipid-anchor, GPI-anchor. HYOU1_HUMAN Hypoxia HYOU1 EPI, ENDO Symptoms Endoplasmic Detection up- reticulum regulated lumen. protein 1 IBP2_HUMAN Insulin-like IGFBP2 LungCancers Secreted. UniProt, growth Literature, factor- Detection, binding Prediction protein 2 IBP3_HUMAN Insulin-like IGFBP3 LungCancers, Secreted. UniProt, growth BenignNodules, Literature, factor- Symptoms Detection, binding Prediction protein 3 ICAM1_HUMAN Intercellular ICAM1 LungCancers, Membrane; UniProt, adhesion BenignNodules, Single-pass Literature, molecule 1 Symptoms type I Detection membrane protein. ICAM3_HUMAN Intercellular ICAM3 EPI, ENDO LungCancers, Membrane; UniProt, adhesion BenignNodules, Single-pass Detection molecule 3 Symptoms type I membrane protein. IDHP_HUMAN Isocitrate IDH2 Secreted, Mitochondrion. Prediction dehydrogenase ENDO [NADP], mitochondrial IF4A1_HUMAN Eukaryotic EIF4A1 Secreted, Detection, initiation EPI, ENDO Prediction factor 4A-I IGF1_HUMAN Insulin-like IGF1 LungCancers, Secreted.|Secreted. UniProt, growth BenignNodules, Literature, factor I Symptoms Detection, Prediction IKIP_HUMAN Inhibitor of IKIP ENDO Symptoms Endoplasmic UniProt, nuclear reticulum Prediction factor membrane; kappa-B Single-pass kinase- membrane interacting protein. protein Note = Isoform 4 deletion of the hydrophobic, or transmembrane region between AA 45-63 results in uniform distribution troughout the cell, suggesting that this region is responsible for endoplasmic reticulum localization. IL18_HUMAN Interleukin- IL18 LungCancers, Secreted. UniProt, 18 BenignNodules, Literature, Symptoms Prediction IL19_HUMAN Interleukin- IL19 LungCancers Secreted. UniProt, 19 Detection, Prediction IL22_HUMAN Interleukin- IL22 LungCancers, Secreted. UniProt, 22 BenignNodules Prediction IL32_HUMAN Interleukin- IL32 LungCancers, Secreted. UniProt, 32 BenignNodules Prediction IL7_HUMAN Interleukin- IL7 LungCancers, Secreted. UniProt, 7 BenignNodules Literature, Prediction IL8_HUMAN Interleukin- IL8 LungCancers, Secreted. UniProt, 8 BenignNodules, Literature Symptoms ILEU_HUMAN Leukocyte SERPINB1 Secreted, Cytoplasm (By Detection, elastase EPI similarity). Prediction inhibitor ILK_HUMAN Integrin- ILK Secreted LungCancers, Cell junction, Literature, linked BenignNodules, focal adhesion. Detection protein Symptoms Cell membrane; kinase Peripheral membrane protein; Cytoplasmic side. INHBA_HUMAN Inhibin INHBA LungCancers, Secreted. UniProt, beta A BenignNodules Literature, chain Prediction ISLR_HUMAN Immunoglobulin ISLR LungCancers Secreted UniProt, superfamily (Potential). Detection, containing Prediction leucine- rich repeat protein ITA5_HUMAN Integrin ITGA5 EPI LungCancers, Membrane; UniProt, alpha-5 BenignNodules, Single-pass Literature, Symptoms type I Detection membrane protein. ITAM_HUMAN Integrin ITGAM EPI, ENDO LungCancers, Membrane; UniProt, alpha-M BenignNodules, Single-pass Literature Symptoms type I membrane protein. K0090_HUMAN Uncharacterized KIAA0090 EPI Symptoms Membrane; UniProt, protein Single-pass Prediction KIAA0090 type I membrane protein (Potential). K1C18_HUMAN Keratin, KRT18 Secreted LungCancers, Cytoplasm, Literature, type I BenignNodules perinuclear Detection, cytoskeletal region. Prediction 18 K1C19_HUMAN Keratin, KRT19 LungCancers, Literature, type I BenignNodules Detection, cytoskeletal Prediction 19 K2C8_HUMAN Keratin, KRT8 EPI LungCancers Cytoplasm. Literature, type II Detection cytoskeletal 8 KIT_HUMAN Mast/stem KIT LungCancers Membrane; UniProt, cell growth Single-pass Literature, factor type I Detection receptor membrane protein. KITH_HUMAN Thymidine TK1 LungCancers Cytoplasm. Literature, kinase, Prediction cytosolic KLK11_HUMAN Kallikrein- KLK11 LungCancers Secreted. UniProt, 11 Literature, Prediction KLK13_HUMAN Kallikrein- KLK13 LungCancers Secreted UniProt, 13 (Probable). Literature, Detection, Prediction KLK14_HUMAN Kallikrein- KLK14 LungCancers, Secreted, UniProt, 14 Symptoms extracellular Literature, space. Prediction KLK6_HUMAN Kallikrein- KLK6 LungCancers, Secreted. UniProt, 6 BenignNodules, Nucleus, Literature, Symptoms nucleolus. Detection, Cytoplasm. Prediction Mitochondrion. Microsome. Note = In brain, detected in the nucleus of glial cells and in the nucleus and cytoplasm of neurons. Detected in the mitochondrial and microsomal fractions of HEK-293 cells and released into the cytoplasm following cell stress. KNG1_HUMAN Kininogen- KNG1 LungCancers, Secreted, UniProt, 1 BenignNodules, extracellular Detection, Symptoms space. Prediction KPYM_HUMAN Pyruvate PKM2 Secreted, LungCancers, Cytoplasm. Literature, kinase EPI Symptoms Nucleus. Detection isozymes Note = Translocates M1/M2 to the nucleus in response to different apoptotic stimuli. Nuclear translocation is sufficient to induce cell death that is caspase independent, isoform-specific and independent of its enzymatic actvity. KRT35_HUMAN Keratin, KRT35 ENDO Detection, type I Prediction cuticular Ha5 LAMB2_HUMAN Laminin LAMB2 ENDO LungCancers, Secreted, UniProt, subunit Symptoms extracellular Detection, beta-2 space, Prediction extracellular matrix, basement membrane. Note = S-laminin is concentrated in the synaptic cleft of the neuromuscular junction. LDHA_HUMAN L-lactate LDHA Secreted, LungCancers Cytoplasm. Literature, dehydrogenase EPI, ENDO Detection, A chain Prediction LDHB_HUMAN L-lactate LDHB EPI LungCancers Cytoplasm. Detection, dehydrogenase Prediction B chain LEG1_HUMAN Galectin-1 LGALS1 Secreted LungCancers Secreted, UniProt, extracellular Detection space, extracellular matrix. LEG3_HUMAN Galectin-3 LGALS3 LungCancers, Nucleus. Literature, BenignNodules Note = Cytoplasmic Detection, in Prediction adenomas and carcinomas. May be secreted by a non- classical secretory pathway and associate with the cell surface. LEG9_HUMAN Galectin-9 LGALS9 ENDO Symptoms Cytoplasm (By UniProt similarity). Secreted (By similarity). Note = May also be secreted by a non-classical secretory pathway (By similarity). LG3BP_HUMAN Galectin-3- LGALS3BP Secreted LungCancers, Secreted. UniProt, binding BenignNodules, Secreted, Literature, protein Symptoms extracellular Detection, space, Prediction extracellular matrix. LPLC3_HUMAN Long C20orf185 LungCancers Secreted (By UniProt, palate, lung similarity). Prediction and nasal Cytoplasm. epithelium Note = According carcinoma- to associated PubMed: protein 3 12837268 it is cytoplasmic. LPLC4_HUMAN Long C20orf186 LungCancers Secreted (By UniProt, palate, lung similarity). Prediction and nasal Cytoplasm. epithelium carcinoma- associated protein 4 LPPRC_HUMAN Leucine- LRPPRC Secreted, LungCancers, Mitochondrion. Prediction rich PPR ENDO Symptoms Nucleus, motif- nucleoplasm. containing Nucleus inner protein, membrane. mitochondrial Nucleus outer membrane. Note = Seems to be predominantly mitochondrial. LRP1_HUMAN Prolow- LRP1 EPI LungCancers, Low-density UniProt, density Symptoms lipoprotein Detection lipoprotein receptor-related receptor- protein 1 85 related kDa subunit: protein 1 Cell membrane; Single-pass type I membrane protein. Membrane, coated pit.|Low- density lipoprotein receptor-related protein 1 515 kDa subunit: Cell membrane; Peripheral membrane protein; Extracellular side. Membrane, coated pit.|Low- density lipoprotein receptor-related protein 1 intracellular domain: Cytoplasm. Nucleus. Note = After cleavage, the intracellular domain (LRPICD) is detected both in the cytoplasm and in the nucleus. LUM_HUMAN Lumican LUM Secreted, LungCancers, Secreted, UniProt, EPI BenignNodules, extracellular Detection, Symptoms space, Prediction extracellular matrix (By similarity). LY6K_HUMAN Lymphocyte LY6K LungCancers, Secreted. UniProt, antigen Symptoms Cytoplasm. Cell Prediction 6K membrane; Lipid- anchor, GPI-anchor (Potential). LYAM2_HUMAN E-selectin SELE LungCancers, Membrane; UniProt, BenignNodules, Single-pass Literature, Symptoms type I Detection membrane protein. LYAM3_HUMAN P-selectin SELP LungCancers, Membrane; UniProt, BenignNodules, Single-pass Literature, Symptoms type I Detection membrane protein. LYOX_HUMAN Protein- LOX LungCancers, Secreted, UniProt, lysine 6- BenignNodules extracellular Detection, oxidase space. Prediction LYPD3_HUMAN Ly6/PLAUR LYPD3 LungCancers Cell membrane; Detection, domain- Lipid-anchor, Prediction containing GPI-anchor. protein 3 MAGA4_HUMAN Melanoma- MAGEA4 LungCancers Literature, associated Prediction antigen 4 MASP1_HUMAN Mannan- MASP1 LungCancers, Secreted. UniProt, binding Symptoms Detection, lectin Prediction serine protease 1 MDHC_HUMAN Malate MDH1 Secreted Cytoplasm. Literature, dehydrogenase, Detection, cytoplasmic Prediction MDHM_HUMAN Malate MDH2 ENDO LungCancers Mitochondrion Detection, dehydrogenase, matrix. Prediction mitochondrial MIF_HUMAN Macrophage MIF Secreted LungCancers, Secreted. UniProt, migration BenignNodules, Cytoplasm. Literature, inhibitory Symptoms Note = Does not Prediction factor have a cleavable signal sequence and is secreted via a specialized, non- classical pathway. Secreted by macrophages upon stimulation by bacterial lipopolysaccharide (LPS), or by M. tuberculosis antigens. MLH1_HUMAN DNA MLH1 ENDO LungCancers, Nucleus. Literature mismatch BenignNodules, repair Symptoms protein Mlh1 MMP1_HUMAN Interstitial MMP1 LungCancers, Secreted, UniProt, collagenase BenignNodules, extracellular Literature, Symptoms space, Prediction extracellular matrix (Probable). MMP11_HUMAN Stromelysin- MMP11 LungCancers, Secreted, UniProt, 3 Symptoms extracellular Literature, space, Prediction extracellular matrix (Probable). MMP12_HUMAN Macrophage MMP12 LungCancers, Secreted, UniProt, metalloelastase BenignNodules, extracellular Literature, Symptoms space, Prediction extracellular matrix (Probable). MMP14_HUMAN Matrix MMP14 ENDO LungCancers, Membrane; UniProt, metalloproteinase- BenignNodules, Single-pass Literature, 14 Symptoms type I Detection membrane protein (Potential). Melanosome. Note = Identified by mass spectrometry in melanosome fractions from stage I to stage IV. MMP2_HUMAN 72 kDa MMP2 LungCancers, Secreted, UniProt, type IV BenignNodules, extracellular Literature, collagenase Symptoms space, Detection, extracellular Prediction matrix (Probable). MMP26_HUMAN Matrix MMP26 LungCancers Secreted, UniProt, metalloproteinase- extracellular Prediction 26 space, extracellular matrix. MMP7_HUMAN Matrilysin MMP7 LungCancers, Secreted, UniProt, BenignNodules, extracellular Literature, Symptoms space, Prediction extracellular matrix (Probable). MMP9_HUMAN Matrix MMP9 LungCancers, Secreted, UniProt, metalloproteinase- BenignNodules, extracellular Literature, 9 Symptoms space, Detection, extracellular Prediction matrix (Probable). MOGS_HUMAN Mannosyl- MOGS ENDO Endoplasmic UniProt, oligosaccharide reticulum Prediction glucosidase membrane; Single-pass type II membrane protein. MPRI_HUMAN Cation- IGF2R EPI, ENDO LungCancers, Lysosome UniProt, independent Symptoms membrane; Literature, mannose-6- Single-pass Detection phosphate type I receptor membrane protein. MRP3_HUMAN Canalicular ABCC3 EPI LungCancers Membrane; Literature, multispecific Multi-pass Detection organic membrane anion protein. transporter 2 MUC1_HUMAN Mucin-1 MUC1 EPI LungCancers, Apical cell UniProt, BenignNodules, membrane; Literature, Symptoms Single-pass Prediction type I membrane protein. Note = Exclusively located in the apical domain of the plasma membrane of highly polarized epithelial cells. After endocytosis, internalized and recycled to the cell membrane. Located to microvilli and to the tips of long filopodial prolusions.|Isoform 5: Secreted.|Isoform 7: Secreted.|Isoform 9: Secreted.|Mucin- 1 subunit beta: Cell membrane. Cytoplasm. Nucleus. Note = On EGF and PDGFRB stimulation, transported to the nucleus through interaction with CTNNB1, a process which is stimulated by phosphorylation. On HRG stimulation, colocalizes with JUP/gamma- catenin at the nucleus. MUC16_HUMAN Mucin-16 MUC16 LungCancers Cell membrane; UniProt, Single-pass Detection type I membrane protein. Secreted, extracellular space. Note = May be liberated into the extracellular space following the phosphorylation of the intracellular C- terminus which induces the proteolytic cleavage and liberation of the extracellular domain. MUC4_HUMAN Mucin-4 MUC4 LungCancers, Membrane; UniProt BenignNodules Single-pass membrane protein (Potential). Secreted. Note = Isoforms lacking the Cys- rich region, EGF-like domains and transmembrane region are secreted. Secretion occurs by splicing or proteolytic processing.|Mucin- 4 beta chain: Cell membrane; Single- pass membrane protein.|Mucin- 4 alpha chain: Secreted.|Isoform 3: Cell membrane; Single-pass membrane protein.|Isoform 15: Secreted. MUC5B_HUMAN Mucin-5B MUC5B LungCancers, Secreted. UniProt, BenignNodules Detection, Prediction MUCL1_HUMAN Mucin-like MUCL1 LungCancers Secreted UniProt, protein 1 (Probable). Prediction Membrane (Probable). NAMPT_HUMAN Nicotinamide NAMPT EPI LungCancers, Cytoplasm (By Literature, phosphoribosyl- BenignNodules, similarity). Detection transferase Symptoms NAPSA_HUMAN Napsin-A NAPSA Secreted LungCancers Prediction NCF4_HUMAN Neutrophil NCF4 ENDO Cytoplasm. Prediction cytosol factor 4 NDKA_HUMAN Nucleoside NME1 Secreted LungCancers, Cytoplasm. Literature, diphosphate BenignNodules, Nucleus. Detection kinase A Symptoms Note = Cell- cycle dependent nuclear localization which can be induced by interaction with Epstein-barr viral proteins or by degradation of the SET complex by GzmA. NDKB_HUMAN Nucleoside NME2 Secreted, BenignNodules Cytoplasm. Literature, diphosphate EPI Nucleus. Detection kinase B Note = Isoform 2 is mainly cytoplasmic and isoform 1 and isoform 2 are excluded from the nucleolus. NDUS1_HUMAN NADH- NDUFS1 Secreted, Symptoms Mitochondrion Prediction ubiquinone ENDO inner oxidoreductase membrane. 75 kDa subunit, mitochondrial NEBL_HUMAN Nebulette NEBL ENDO Prediction NEK4_HUMAN Serine/threonine- NEK4 ENDO LungCancers Nucleus Prediction protein (Probable). kinase Nek4 NET1_HUMAN Netrin-1 NTN1 LungCancers, Secreted, UniProt, BenignNodules extracellular Literature, space, Prediction extracellular matrix (By similarity). NEU2_HUMAN Vasopressin- AVP LungCancers, Secreted. UniProt, neurophysin Symptoms Prediction 2- copeptin NGAL_HUMAN Neutrophil LCN2 EPI LungCancers, Secreted. UniProt, gelatinase- BenignNodules, Detection, associated Symptoms Prediction lipocalin NGLY1_HUMAN Peptide- NGLY1 ENDO Cytoplasm. Detection, N(4)-(N- Prediction acetyl-beta- glucosaminyl)asparagine amidase NHRF1_HUMAN Na(+)/H(+) SLC9A3R1 EPI BenignNodules Endomembrane Detection exchange system; regulatory Peripheral cofactor membrane NHE-RF1 protein. Cell projection, filopodium. Cell projection, ruffle. Cell projection, microvillus. Note = Colocalizes with actin in microvilli-rich apical regions of the syncytiotrophoblast. Found in microvilli, ruffling membrane and filopodia of HeLa cells. Present in lipid rafts of T-cells. NIBAN_HUMAN Protein FAM129A EPI Cytoplasm. Literature, Niban Detection NMU_HUMAN Neuromedin- NMU LungCancers Secreted. UniProt, U Prediction NRP1_HUMAN Neuropilin- NRP1 LungCancers, Cell membrane; UniProt, 1 BenignNodules, Single-pass Literature, Symptoms type I Detection, membrane Prediction protein.|Isoform 2: Secreted. ODAM_HUMAN Odontogenic ODAM LungCancers Secreted (By UniProt, ameloblast- similarity). Prediction associated protein OSTP_HUMAN Osteopontin SPP1 LungCancers, Secreted. UniProt, BenignNodules, Literature, Symptoms Detection, Prediction OVOS2_HUMAN Ovostatin OVOS2 ENDO Secreted (By UniProt, homolog 2 similarity). Prediction P5CS_HUMAN Delta-1- ALDH18A1 ENDO Mitochondrion Prediction pyrroline- inner 5- membrane. carboxylate synthase PA2GX_HUMAN Group 10 PLA2G10 Symptoms Secreted. UniProt secretory phospholipase A2 PAPP1_HUMAN Pappalysin- PAPPA LungCancers, Secreted. UniProt, 1 BenignNodules, Literature, Symptoms Prediction PBIP1_HUMAN Pre-B-cell PBXIP1 EPI Cytoplasm, Prediction leukemia cytoskeleton. transcription Nucleus. factor- Note = Shuttles interacting between the protein 1 nucleus and the cytosol. Mainly localized in the cytoplasm, associated with microtubules. Detected in small amounts in the nucleus. PCBP1_HUMAN Poly(rC)- PCBP1 EPI, ENDO Nucleus. Detection, binding Cytoplasm. Prediction protein 1 Note = Loosely bound in the nucleus. May shuttle between the nucleus and the cytoplasm. PCBP2_HUMAN Poly(rC)- PCBP2 EPI Nucleus. Detection, binding Cytoplasm. Prediction protein 2 Note = Loosely bound in the nucleus. May shuttle between the nucleus and the cytoplasm. PCD15_HUMAN Protocadherin- PCDH15 ENDO Cell membrane; UniProt, 15 Single-pass Detection type I membrane protein (By similarity).|Isoform 3: Secreted. PCNA_HUMAN Proliferating PCNA EPI LungCancers, Nucleus. Literature, cell BenignNodules, Prediction nuclear Symptoms antigen PCYOX_HUMAN Prenylcysteine PCYOX1 Secreted LungCancers, Lysosome. Detection, oxidase Symptoms Prediction 1 PDGFA_HUMAN Platelet- PDGFA LungCancers Secreted. UniProt, derived Literature, growth Prediction factor subunit A PDGFB_HUMAN Platelet- PDGFB LungCancers, Secreted. UniProt, derived BenignNodules, Literature, growth Symptoms Detection, factor Prediction subunit B PDGFD_HUMAN Platelet- PDGFD LungCancers Secreted. UniProt, derived Prediction growth factor D PDIA3_HUMAN Protein PDIA3 ENDO LungCancers Endoplasmic Detection, disulfide- reticulum Prediction isomerase lumen (By A3 similarity). Melanosome. Note = Identified by mass spectrometry in melanosome fractions from stage I to stage IV. PDIA4_HUMAN Protein PDIA4 Secreted, Endoplasmic Detection, disulfide- EPI, ENDO reticulum Prediction isomerase lumen. A4 Melanosome. Note = Identified by mass spectrometry in melanosome fractions from stage I to stage IV. PDIA6_HUMAN Protein PDIA6 Secreted, Endoplasmic Detection, disulfide- EPI, ENDO reticulum Prediction isomerase lumen (By A6 similarity). Melanosome. Note = Identified by mass spectrometry in melanosome fractions from stage I to stage IV. PECA1_HUMAN Platelet PECAM1 LungCancers, Membrane; UniProt, endothelial BenignNodules, Single-pass Literature, cell Symptoms type I Detection adhesion membrane molecule protein. PEDF_HUMAN Pigment SERPINF1 LungCancers, Secreted. UniProt, epithelium- Symptoms Melanosome. Literature, derived Note = Enriched Detection, factor in stage I Prediction melanosomes. PERM_HUMAN Myeloperoxidase MPO Secreted, LungCancers, Lysosome. Literature, EPI, ENDO BenignNodules, Detection, Symptoms Prediction PERP1_HUMAN Plasma PACAP EPI, ENDO Secreted UniProt, cell- (Potential). Detection, induced Cytoplasm. Prediction resident Note = In endoplasmic (PubMed: reticulum 11350957) protein diffuse granular localization in the cytoplasm surrounding the nucleus. PGAM1_HUMAN Phosphoglycerate PGAM1 Secreted, LungCancers, Detection mutase 1 EPI Symptoms PLAC1_HUMAN Placenta- PLAC1 LungCancers Secreted UniProt, specific (Probable). Prediction protein 1 PLACL_HUMAN Placenta- PLAC1L LungCancers Secreted UniProt, specific 1- (Potential). Prediction like protein PLIN2_HUMAN Perilipin-2 ADFP ENDO LungCancers Membrane; Prediction Peripheral membrane protein. PLIN3_HUMAN Perilipin-3 M6PRBP1 EPI Cytoplasm. Detection, Endosome Prediction membrane; Peripheral membrane protein; Cytoplasmic side (Potential). Lipid droplet (Potential). Note = Membrane associated on endosomes. Detected in the envelope and the core of lipid bodies and in lipid sails. PLOD1_HUMAN Procollagen- PLOD1 EPI, ENDO Rough Prediction lysine, 2- endoplasmic oxoglutarate reticulum 5- membrane; dioxygenase Peripheral 1 membrane protein; Lumenal side. PLOD2_HUMAN Procollagen- PLOD2 ENDO BenignNodules, Rough Prediction lysine, 2- Symptoms endoplasmic oxoglutarate reticulum 5- membrane; dioxygenase Peripheral 2 membrane protein; Lumenal side. PLSL_HUMAN Plastin-2 LCP1 Secreted, LungCancers Cytoplasm, Detection, EPI cytoskeleton. Prediction Cell junction. Cell projection. Cell projection, ruffle membrane; Peripheral membrane protein; Cytoplasmic side (By similarity). Note = Relocalizes to the immunological synapse between peripheral blood T lymphocytes and antibody- presenting cells in response to costimulation through TCR/CD3 and CD2 or CD28. Associated with the actin cytoskeleton at membrane ruffles (By similarity). Relocalizes to actin-rich cell projections upon serine phosphorylation. PLUNC_HUMAN Protein PLUNC LungCancers, Secreted (By UniProt, Plunc BenignNodules similarity). Prediction Note = Found in the nasal mucus (By similarity). Apical side of airway epithelial cells. Detected in nasal mucus (By similarity). PLXB3_HUMAN Plexin-B3 PLXNB3 ENDO Membrane; UniProt, Single-pass Detection, type I Prediction membrane protein. PLXC1_HUMAN Plexin-C1 PLXNC1 EPI Membrane; UniProt, Single-pass Detection type I membrane protein (Potential). POSTN_HUMAN Periostin POSTN Secreted, LungCancers, Secreted, UniProt, ENDO BenignNodules, extracellular Literature, Symptoms space, Detection, extracellular Prediction matrix. PPAL_HUMAN Lysosomal ACP2 EPI Symptoms Lysosome UniProt, acid membrane; Prediction phosphatase Single-pass membrane protein; Lumenal side. Lysosome lumen. Note = The soluble form arises by proteolytic processing of the membrane- bound form. PPBT_HUMAN Alkaline ALPL EPI LungCancers, Cell membrane; Literature, phosphatase, BenignNodules, Lipid-anchor, Detection, tissue- Symptoms GPI-anchor. Prediction nonspecific isozyme PPIB_HUMAN Peptidyl- PPIB Secreted, Endoplasmic Detection, prolyl cis- EPI, ENDO reticulum Prediction trans lumen. isomerase Melanosome. B Note = Identified by mass spectrometry in melanosome fractions from stage I to stage IV. PRDX1_HUMAN Peroxiredoxin- PRDX1 EPI LungCancers Cytoplasm. Detection, 1 Melanosome. Prediction Note = Identified by mass spectrometry in melanosome fractions from stage I to stage IV. PRDX4_HUMAN Peroxiredoxin- PRDX4 Secreted, Cytoplasm. Literature, 4 EPI, ENDO Detection, Prediction PROF1_HUMAN Profilin-1 PFN1 Secreted, LungCancers Cytoplasm, Detection EPI cytoskeleton. PRP31_HUMAN U4/U6 PRPF31 ENDO Nucleus Prediction small speckle. nuclear Nucleus, Cajal ribonucleoprotein body. Prp31 Note = Predominantly found in speckles and in Cajal bodies. PRS6A_HUMAN 26S PSMC3 EPI BenignNodules Cytoplasm Detection protease (Potential). regulatory Nucleus subunit 6A (Potential). PSCA_HUMAN Prostate PSCA LungCancers Cell membrane; Literature, stem cell Lipid-anchor, Prediction antigen GPI-anchor. PTGIS_HUMAN Prostacyclin PTGIS EPI LungCancers, Endoplasmic UniProt, synthase BenignNodules reticulum Detection, membrane; Prediction Single-pass membrane protein. PTPA_HUMAN Serine/threonine- PPP2R4 ENDO Symptoms Detection, protein Prediction phosphatase 2A activator PTPRC_HUMAN Receptor- PTPRC Secreted, LungCancers Membrane; UniProt, type EPI, ENDO Single-pass Detection, tyrosine- type I Prediction protein membrane phosphatase protein. C PTPRJ_HUMAN Receptor- PTPRJ EPI LungCancers, Membrane; UniProt, type Symptoms Single-pass Detection, tyrosine- type I Prediction protein membrane phosphatase protein. eta PVR_HUMAN Poliovirus PVR Symptoms Isoform Alpha: UniProt, receptor Cell membrane; Detection, Single-pass Prediction type I membrane protein.|Isoform Delta: Cell membrane; Single-pass type I membrane protein.|Isoform Beta: Secreted.|Isoform Gamma: Secreted. RAB32_HUMAN Ras-related RAB32 EPI Mitochondrion. Prediction protein Rab-32 RAGE_HUMAN Advanced AGER Secreted LungCancers, Isoform 1: Cell UniProt, glycosylation BenignNodules membrane; Literature end Single-pass product- type I specific membrane receptor protein.|Isoform 2: Secreted. RAN_HUMAN GTP- RAN Secreted, LungCancers, Nucleus. Detection, binding EPI BenignNodules Cytoplasm. Prediction nuclear Melanosome. protein Ran Note = Becomes dispersed throughout the cytoplasm during mitosis. Identified by mass spectrometry in melanosome fractions from stage I to stage IV. RAP2B_HUMAN Ras-related RAP2B EPI Cell membrane; Prediction protein Lipid-anchor; Rap-2b Cytoplasmic side (Potential). RAP2C_HUMAN Ras-related RAP2C EPI Cell membrane; Prediction protein Lipid-anchor; Rap-2c Cytoplasmic side (Potential). RCN3_HUMAN Reticulocalbin- RCN3 EPI Symptoms Endoplasmic Prediction 3 reticulum lumen (Potential). RL24_HUMAN 60S RPL24 EPI Prediction ribosomal protein L24 S10A1_HUMAN Protein S100A1 Symptoms Cytoplasm. Literature, S100-A1 Prediction S10A6_HUMAN Protein S100A6 Secreted LungCancers Nucleus Literature, S100-A6 envelope. Detection, Cytoplasm. Prediction S10A7_HUMAN Protein S100A7 LungCancers Cytoplasm. UniProt, S100-A7 Secreted. Literature, Note = Secreted Detection, by a non- Prediction classical secretory pathway. SAA_HUMAN Serum SAA1 Symptoms Secreted. UniProt, amyloid A Literature, protein Detection, Prediction SCF_HUMAN Kit ligand KITLG LungCancers, Isoform 1: Cell UniProt, Symptoms membrane; Literature Single-pass type I membrane protein (By similarity). Secreted (By similarity). Note = Also exists as a secreted soluble form (isoform 1 only) (By similarity).|Isoform 2: Cell membrane; Single-pass type I membrane protein (By similarity). Cytoplasm, cytoskeleton (By similarity). SDC1_HUMAN Syndecan-1 SDC1 LungCancers, Membrane; UniProt, BenignNodules, Single-pass Literature, Symptoms type I Detection membrane protein. SEM3G_HUMAN Semaphorin- SEMA3G LungCancers Secreted (By UniProt, 3G similarity). Prediction SEPR_HUMAN Seprase FAP ENDO Symptoms Cell membrane; UniProt, Single-pass Literature, type II Detection membrane protein. Cell projection, lamellipodium membrane; Single-pass type II membrane protein. Cell projection, invadopodium membrane; Single-pass type II membrane protein. Note = Found in cell surface lamellipodia, invadopodia and on shed vesicles. SERPH_HUMAN Serpin H1 SERPINH1 Secreted, LungCancers, Endoplasmic Detection, EPI, ENDO BenignNodules reticulum Prediction lumen. SFPA2_HUMAN Pulmonary SFTPA2 Secreted LungCancers, Secreted, UniProt, surfactant- BenignNodules extracellular Prediction associated space, protein A2 extracellular matrix. Secreted, extracellular space, surface film. SFTA1_HUMAN Pulmonary SFTPA1 Secreted LungCancers, Secreted, UniProt, surfactant- BenignNodules, extracellular Prediction associated Symptoms space, protein A1 extracellular matrix. Secreted, extracellular space, surface film. SG3A2_HUMAN Secretoglobin SCGB3A2 LungCancers, Secreted. UniProt, family BenignNodules Prediction 3A member 2 SGPL1_HUMAN Sphingosine- SGPL1 ENDO Endoplasmic UniProt, 1- reticulum Prediction phosphatelyase membrane; 1 Single-pass type III membrane protein. SIAL_HUMAN Bone IBSP LungCancers Secreted. UniProt, sialoprotein Literature, 2 Prediction SLPI_HUMAN Antileuko- SLPI LungCancers, Secreted. UniProt, proteinase BenignNodules Literature, Detection, Prediction SMD3_HUMAN Small SNRPD3 Secreted BenignNodules Nucleus. Prediction nuclear ribonucleoprotein SmD3 SMS_HUMAN Somatostatin SST LungCancers Secreted. UniProt, Literature, Prediction SODM_HUMAN Superoxide SOD2 Secreted LungCancers, Mitochondrion Literature, dismutase BenignNodules, matrix. Detection, [Mn], Symptoms Prediction mitochondrial SORL_HUMAN Sortilin- SORL1 EPI LungCancers, Membrane; UniProt, related Symptoms Single-pass Detection receptor type I membrane protein (Potential). SPB3_HUMAN Serpin B3 SERPINB3 LungCancers, Cytoplasm. Literature, BenignNodules Note = Seems to Detection also be secreted in plasma by cancerous cells but at a low level. SPB5_HUMAN Serpin B5 SERPINB5 LungCancers Secreted, UniProt, extracellular Detection space. SPON2_HUMAN Spondin-2 SPON2 LungCancers, Secreted, UniProt, BenignNodules extracellular Prediction space, extracellular matrix (By similarity). SPRC_HUMAN SPARC SPARC LungCancers, Secreted, UniProt, BenignNodules, extracellular Literature, Symptoms space, Detection, extracellular Prediction matrix, basement membrane. Note = In or around the basement membrane. SRC_HUMAN Proto- SRC ENDO LungCancers, Literature oncogene BenignNodules, tyrosine- Symptoms protein kinase Src SSRD_HUMAN Translocon- SSR4 Secreted, Endoplasmic UniProt, associated ENDO reticulum Prediction protein membrane; subunit Single-pass delta type I membrane protein. STAT1_HUMAN Signal STAT1 EPI LungCancers, Cytoplasm. Detection transducer BenignNodules Nucleus. and Note = Translocated activator of into the transcription nucleus in 1- response to alpha/beta IFN-gamma- induced tyrosine phosphorylation and dimerization. STAT3_HUMAN Signal STAT3 ENDO LungCancers, Cytoplasm. Prediction transducer BenignNodules, Nucleus. and Symptoms Note = Shuttles activator of between the transcription nucleus and the 3 cytoplasm. Constitutive nuclear presence is independent of tyrosine phosphorylation. STC1_HUMAN Stanniocalcin-1 STC1 LungCancers, Secreted. UniProt, Symptoms Prediction STT3A_HUMAN Dolichyl- STT3A EPI Symptoms Endoplasmic Literature diphospho- reticulum oligosaccharide-- membrane; protein Multi-pass glycosyltransferase membrane subunit protein. STT3A TAGL_HUMAN Transgelin TAGLN EPI LungCancers Cytoplasm Literature, (Probable). Prediction TARA_HUMAN TRIO and TRIOBP ENDO Nucleus. Detection, F-actin- Cytoplasm, Prediction binding cytoskeleton. protein Note = Localized to F-actin in a periodic pattern. TBA1B_HUMAN Tubulin TUBA1B EPI LungCancers Detection alpha-1B chain TBB2A_HUMAN Tubulin TUBB2A EPI LungCancers, Detection, beta-2A BenignNodules Prediction chain TBB3_HUMAN Tubulin TUBB3 EPI LungCancers, Detection beta-3 BenignNodules chain TBB5_HUMAN Tubulin TUBE EPI LungCancers, Detection beta chain BenignNodules TCPA_HUMAN T-complex TCP1 EPI Cytoplasm. Prediction protein 1 subunit alpha TCPD_HUMAN T-complex CCT4 EPI Cytoplasm. Detection, protein 1 Melanosome. Prediction subunit Note = Identified delta by mass spectrometry in melanosome fractions from stage I to stage IV. TCPQ_HUMAN T-complex CCT8 Secreted, Cytoplasm. Prediction protein 1 EPI subunit theta TCPZ_HUMAN T-complex CCT6A Secreted, Cytoplasm. Detection protein 1 EPI subunit zeta TDRD3_HUMAN Tudor TDRD3 ENDO Cytoplasm. Prediction domain- Nucleus. containing Note = Predominantly protein 3 cytoplasmic. Associated with actively translating polyribosomes and with mRNA stress granules. TENA_HUMAN Tenascin TNC ENDO LungCancers, Secreted, UniProt, BenignNodules, extracellular Literature, Symptoms space, Detection extracellular matrix. TENX_HUMAN Tenascin-X TNXB ENDO LungCancers, Secreted, UniProt, Symptoms extracellular Detection, space, Prediction extracellular matrix. TERA_HUMAN Transitional VCP EPI LungCancers, Cytoplasm, Detection endoplasmic BenignNodules cytosol. reticulum Nucleus. ATPase Note = Present in the neuronal hyaline inclusion bodies specifically found in motor neurons from amyotrophic lateral sclerosis patients. Present in the Lewy bodies specifically found in neurons from Parkinson disease patients. TETN_HUMAN Tetranectin CLEC3B LungCancers Secreted. UniProt, Literature, Detection, Prediction TF_HUMAN Tissue F3 LungCancers, Membrane; UniProt, factor BenignNodules, Single-pass Literature Symptoms type I membrane protein. TFR1_HUMAN Transferrin TFRC Secreted, LungCancers, Cell membrane; UniProt, receptor EPI, ENDO BenignNodules, Single-pass Literature, protein 1 Symptoms type II Detection membrane protein. Melanosome. Note = Identified by mass spectrometry in melanosome fractions from stage I to stage IV.|Transferrin receptor protein 1, serum form: Secreted. TGFA_HUMAN Protransforming TGFA LungCancers, Transforming UniProt, growth BenignNodules growth factor Literature factor alpha alpha: Secreted, extracellular space.|Protransforming growth factor alpha: Cell membrane; Single-pass type I membrane protein. THAS_HUMAN Thromboxane-A TBXAS1 EPI, ENDO LungCancers, Membrane; Prediction synthase BenignNodules, Multi-pass Symptoms membrane protein. THY1_HUMAN Thy-1 THY1 EPI Symptoms Cell membrane; Detection, membrane Lipid-anchor, Prediction glycoprotein GPI-anchor (By similarity). TIMP1_HUMAN Metalloproteinase TIMP1 LungCancers, Secreted. UniProt, inhibitor 1 BenignNodules, Literature, Symptoms Detection, Prediction TIMP3_HUMAN Metalloproteinase TIMP3 LungCancers, Secreted, UniProt, inhibitor 3 BenignNodules extracellular Literature, space, Prediction extracellular matrix. TLL1_HUMAN Tolloid- TLL1 ENDO Secreted UniProt, like protein (Probable). Prediction 1 TNF12_HUMAN Tumor TNFSF12 LungCancers, Cell membrane; UniProt necrosis BenignNodules Single-pass factor type II ligand membrane superfamily protein.|Tumor member 12 necrosis factor ligand superfamily member 12, secreted form: Secreted. TNR6_HUMAN Tumor FAS LungCancers, Isoform 1: Cell UniProt, necrosis BenignNodules, membrane; Literature, factor Symptoms Single-pass Prediction receptor type I superfamily membrane member 6 protein.|Isoform 2: Secreted.|Isoform 3: Secreted.|Isoform 4: Secreted.|Isoform 5: Secreted.|Isoform 6: Secreted. TPIS_HUMAN Triosephosphate TPI1 Secreted, Symptoms Literature, isomerase EPI Detection, Prediction TRFL_HUMAN Lactotransferrin LTF Secreted, LungCancers, Secreted. UniProt, EPI, ENDO BenignNodules, Literature, Symptoms Detection, Prediction TSP1_HUMAN Thrombospondin-1 THBS1 LungCancers, Literature, BenignNodules, Detection, Symptoms Prediction TTHY_HUMAN Transthyretin TTR LungCancers, Secreted. UniProt, BenignNodules Cytoplasm. Literature, Detection, Prediction TYPH_HUMAN Thymidine TYMP EPI LungCancers, Literature, phosphorylase BenignNodules, Detection, Symptoms Prediction UGGG1_HUMAN UDP- UGGT1 Secreted, Endoplasmic Detection, glucose: glycoprotein ENDO reticulum Prediction glucosyltransferase lumen. 1 Endoplasmic reticulum-Golgi intermediate compartment. UGGG2_HUMAN UDP- UGGT2 ENDO Endoplasmic Prediction glucose: glycoprotein reticulum glucosyltransferase lumen. 2 Endoplasmic reticulum-Golgi intermediate compartment. UGPA_HUMAN UTP-- UGP2 EPI Symptoms Cytoplasm. Detection glucose-1- phosphate uridylyltransferase UPAR_HUMAN Urokinase PLAUR LungCancers, Isoform 1: Cell UniProt, plasminogen BenignNodules, membrane; Literature, activator Symptoms Lipid-anchor, Prediction surface GPI- receptor anchor.|Isoform 2: Secreted (Probable). UTER_HUMAN Uteroglobin SCGB1A1 LungCancers, Secreted. UniProt, BenignNodules, Literature, Symptoms Detection, Prediction VA0D1_HUMAN V-type ATP6V0D1 EPI Prediction proton ATPase subunit d 1 VAV3_HUMAN Guanine VAV3 ENDO Prediction nucleotide exchange factor VAV3 VEGFA_HUMAN Vascular VEGFA LungCancers, Secreted. UniProt, endothelial BenignNodules, Note = VEGF121 Literature, growth Symptoms is acidic and Prediction factor A freely secreted. VEGF165 is more basic, has heparin-binding properties and, although a signicant proportion remains cell- associated, most is freely secreted. VEGF189 is very basic, it is cell-associated after secretion and is bound avidly by heparin and the extracellular matrix, although it may be released as a soluble form by heparin, heparinase or plasmin. VEGFC_HUMAN Vascular VEGFC LungCancers, Secreted. UniProt, endothelial BenignNodules Literature, growth Prediction factor C VEGFD_HUMAN Vascular FIGF LungCancers Secreted. UniProt, endothelial Literature, growth Prediction factor D VGFR1_HUMAN Vascular FLT1 LungCancers, Isoform Flt1: UniProt, endothelial BenignNodules, Cell membrane; Literature, growth Symptoms Single-pass Detection, factor type I Prediction receptor 1 membrane protein.|Isoform sFlt1: Secreted. VTNC_HUMAN Vitronectin VTN ENDO Symptoms Secreted, UniProt, extracellular Literature, space. Detection, Prediction VWC2_HUMAN Brorin VWC2 LungCancers Secreted, UniProt, extracellular Prediction space, extracellular matrix, basement membrane (By similarity). WNT3A_HUMAN Protein WNT3A LungCancers, Secreted, UniProt, Wnt-3a Symptoms extracellular Prediction space, extracellular matrix. WT1_HUMAN Wilms WT1 LungCancers, Nucleus. Literature, tumor BenignNodules, Cytoplasm (By Prediction protein Symptoms similarity). Note = Shuttles between nucleus and cytoplasm (By similarity).|Isoform 1: Nucleus speckle.|Isoform 4: Nucleus, nucleoplasm. ZA2G_HUMAN Zinc-alpha- AZGP1 LungCancers, Secreted. UniProt, 2- Symptoms Literature, glycoprotein Detection, Prediction ZG16B_HUMAN Zymogen ZG16B LungCancers Secreted UniProt, granule (Potential). Prediction protein 16 homolog B SRM Assay

SRM assays for 388 targeted proteins were developed based on synthetic peptides, using a protocol similar to those described in the literature (Lange, Picotti et al. 2008, Picotti, Rinner et al. 2010, Huttenhain, Soste et al. 2012). Up to five SRM suitable peptides per protein were identified from public sources such as the PeptideAtlas, Human Plasma Proteome Database or by proteotypic prediction tools (Mallick, Schirle et al. 2007) and synthesized. SRM triggered MS/MS spectra were collected on an ABSciex 5500 QTrap for both doubly and triply charged precursor ions. The obtained MS/MS spectra were assigned to individual peptides using MASCOT (cutoff score ≥15) (Perkins, Pappin et al. 1999). Up to four transitions per precursor ion were selected for optimization. The resulting corresponding optimal retention time, declustering potential and collision energy were assembled for all transitions. Optimal transitions were measured on a mixture of all synthetic peptides, a pooled sample of benign patients and a pooled sample of cancer patients. Transitions were analyzed in batches, each containing up to 1750 transitions. Both biological samples were immuno-depleted and digested by trypsin. All three samples were analyzed on an ABSciex 5500 QTrap coupled with a reversed-phase (RP) high-performance liquid chromatography (HPLC) system. The obtained SRM data were manually reviewed to select the two best peptides per protein and the two best transitions per peptide. Transitions having interference with other transitions were not selected. Ratios between intensities of the two best transitions of peptides in the synthetic peptide mixture were also used to assess the specificity of the transitions in the biological samples. The intensity ratio was considered as an important metric defining the SRM assays. The complete transition table is shown below in Table 2.

Lengthy table referenced here US10534002-20200114-T00001 Please refer to the end of the specification for access instructions. Exemplary Protein Detection

The following 164 proteins and their peptides were detected simultaneously in a large-scale experiment of 158 samples using the MS-LC-SRM-MS system described herein.

TABLE 3 SEQ ID Protein Peptide Protein Peptide NO: Detection Detection 1433E_HUMAN EDLVYQAK 7 16 16 1433E_HUMAN IISSIEQK 9 16 0 1433T_HUMAN AVTEQGAELSNEER 16 127 0 1433T_HUMAN TAFDEAIAELDTLNEDSYK 19 127 127 1433Z_HUMAN FLIPNASQAESK 21 157 157 1433Z_HUMAN SVTEQGAELSNEER 23 157 0 6PGD_HUMAN AGQAVDDFIEK 25 90 0 6PGD_HUMAN LVPLLDTGDIIIDGGNSEYR 27 90 90 A1AG1_HUMAN WFYIASAFR 32 157 0 A1AG1_HUMAN YVGGQEHFAHLLILR 33 157 157 ABCD1_HUMAN DAGIALLSITHRPSLWK 34 9 0 ABCD1_HUMAN GLQAPAGEPTQEASGVAAAK 36 9 0 ABCD1_HUMAN NLLTAAADAIER 37 9 9 ADML_HUMAN LAHQIYQFTDK 44 27 27 ADML_HUMAN SPEDSSPDAAR 45 27 0 AIFM1_HUMAN ELWFSDDPNVTK 53 158 158 AIFM1_HUMAN GVIFYLR 54 158 0 ALDOA_HUMAN ADDGRPFPQVIK 57 158 141 ALDOA_HUMAN ALQASALK 58 158 17 AMPN_HUMAN ALEQALEK 62 158 158 AMPN_HUMAN DHSAIPVINR 63 158 0 APOA1_HUMAN AKPALEDLR 78 158 158 APOA1_HUMAN ATEHLSTLSEK 79 158 0 APOE_HUMAN AATVGSLAGQPLQER 82 158 158 APOE_HUMAN LGPLVEQGR 87 158 0 BGH3_HUMAN LTLLAPLNSVFK 139 158 0 BGH3_HUMAN SPYQLVLQHSR 140 158 158 BST1_HUMAN GEGTSAHLR 149 157 0 BST1_HUMAN GFFADYEIPNLQK 150 157 157 C163A_HUMAN INPASLDK 153 158 11 C163A_HUMAN LEVFYNGAWGTVGK 154 158 49 C163A_HUMAN TSYQVYSK 155 158 98 CALU_HUMAN EQFVEFR 172 120 120 CALU_HUMAN TFDQLTPEESK 174 120 0 CATB_HUMAN LPASFDAR 188 62 62 CATB_HUMAN TDQYWEK 190 62 0 CATG_HUMAN NVNPVALPR 192 14 0 CATG_HUMAN SSGVPPEVFTR 193 14 14 CBPB2_HUMAN DTGTYGFLLPER 198 158 158 CBPB2_HUMAN EAFAAVSK 199 158 0 CD14_HUMAN ATVNPSAPR 207 158 0 CD14_HUMAN SWLAELQQWLKPGLK 214 158 158 CD44_HUMAN FAGVFHVEK 227 158 158 CD44_HUMAN YGFIEGHVVIPR 231 158 0 CD59_HUMAN AGLQVYNK 232 156 156 CD59_HUMAN TVLLLVTPFLAAAWSLHP 233 156 0 CDCP1_HUMAN EEGVFTVTPDTK 239 157 0 CDCP1_HUMAN LSLVLVPAQK 241 157 157 CEAM8_HUMAN LFIPNITTK 256 79 79 CEAM8_HUMAN TLTLLSVTR 257 79 0 CERU_HUMAN GAYPLSIEPIGVR 258 158 0 CERU_HUMAN GPEEEHLGILGPVIWAEVGDTIR 259 158 158 CERU_HUMAN NNEGTYYSPNYNPQSR 261 158 0 CH10_HUMAN GGEIQPVSVK 265 158 0 CH10_HUMAN VLLPEYGGTK 266 158 158 CLIC1_HUMAN FSAYIK 288 137 8 CLIC1_HUMAN LAALNPESNTAGLDIFAK 290 137 129 CLIC1_HUMAN NSNPALNDNLEK 291 137 0 CLUS_HUMAN ASSIIDELFQDR 293 158 0 CLUS_HUMAN EIQNAVNGVK 294 158 158 CNTN1_HUMAN AHSDGGDGVVSQVK 303 158 157 CNTN1_HUMAN DGEYVVEVR 304 158 1 CO6A3_HUMAN IGDLHPQIVNLLK 319 158 0 CO6A3_HUMAN VAVVQYSDR 321 158 158 CO6A3_HUMAN WYYDPNTK 322 158 0 COF1_HUMAN EILVGDVGQTVDDPYATFVK 328 127 0 COF1_HUMAN LGGSAVISLEGKPL 329 127 0 COF1_HUMAN YALYDATYETK 330 127 127 COIA1_HUMAN AVGLAGTFR 332 158 37 COIA1_HUMAN TEAPSATGQASSLLGGR 335 158 121 CRP_HUMAN APLTKPLK 341 153 21 CRP_HUMAN ESDTSYVSLK 342 153 132 CRP_HUMAN YEVQGEVFTKPQLWP 343 153 0 CSF1_HUMAN FNSVPLTDTGHER 351 134 113 CSF1_HUMAN ISSLRPQGLSNPSTLSAQPQLSR 352 134 21 CYTB_HUMAN SQLEEK 372 100 0 CYTB_HUMAN SQVVAGTNYFIK 373 100 100 DESP_HUMAN YGDGIQLTR 384 131 131 DMKN_HUMAN QVPGFGVADALGNR 395 128 0 DMKN_HUMAN VSEALGQGTR 397 128 128 DSG2_HUMAN GQIIGNFQAFDEDTGLPAHAR 404 158 1 DSG2_HUMAN ILDVNDNIPVVENK 405 158 157 EF1A1_HUMAN IGGIGTVPVGR 423 158 158 EF1A1_HUMAN QTVAVGVIK 426 158 0 EF2_HUMAN FSVSPVVR 439 125 125 EF2_HUMAN GVQYLNEIK 441 125 0 ENOA_HUMAN AVEHINK 452 156 0 ENOA_HUMAN YISPDQLADLYK 455 156 156 ENOA_HUMAN YNQLLR 456 156 0 ENPL_HUMAN SGTSEFLNK 469 158 1 ENPL_HUMAN SGYLLPDTK 470 158 157 EPHB6_HUMAN RPHFDQLVAAFDK 472 157 0 EPHB6_HUMAN WAAPEVIAHGK 476 157 157 ERBB3_HUMAN GESIEPLDPSEK 483 105 0 ERBB3_HUMAN LAEVPDLLEK 484 105 105 EREG_HUMAN VAQVSITK 487 115 115 EREG_HUMAN VTSGDPELPQV 488 115 0 ERO1A_HUMAN AVLQVVTK 489 121 0 ERO1A_HUMAN LLESDYFR 491 121 0 ERO1A_HUMAN NLLQNIH 492 121 0 ERO1A_HUMAN VLPFFERPDFQLFTGNK 493 121 121 F10A1_HUMAN AIDLFTDAIK 501 35 0 F10A1_HUMAN LQKPNAAIR 503 35 35 FAM3C_HUMAN GINVALANGK 505 97 88 FAM3C_HUMAN SALDTAAR 507 97 9 FAM3C_HUMAN TGEVLDTK 509 97 0 FCGR1_HUMAN HLEEELK 517 39 0 FCGR1_HUMAN VFTEGEPLALR 519 39 39 FIBA_HUMAN GGSTSYGTGSETESPR 554 147 108 FIBA_HUMAN NSLFEYQK 556 147 39 FINC_HUMAN SYTITGLQPGTDYK 561 154 135 FINC_HUMAN VPGTSTSATLTGLTR 562 154 19 FKB11_HUMAN ANYWLK 565 23 0 FKB11_HUMAN DPLVIELGQK 566 23 23 FOLH1_HUMAN GVILYSDPADYFAPGVK 569 138 0 FOLH1_HUMAN LGSGNDFEVFFQR 570 138 138 FRIL_HUMAN DDVALEGVSHFFR 594 151 0 FRIL_HUMAN LGGPEAGLGEYLFER 596 151 151 G3P_HUMAN GALQNIIPASTGAAK 599 150 149 G3P_HUMAN LISWYDNEFGYSNR 600 150 1 G6PD_HUMAN DGLLPENTFIVGYAR 603 43 43 G6PD_HUMAN GGYFDEFGIIR 604 43 0 G6PI_HUMAN AVLHVALR 608 39 6 G6PI_HUMAN TLAQLNPESSLFIIASK 610 39 33 GDIR2_HUMAN DIVSGLK 629 158 158 GDIR2_HUMAN LNYKPPPQK 630 158 0 GELS_HUMAN AQPVQVAEGSEPDGFWEALGGK 634 158 0 GELS_HUMAN TASDFITK 637 158 158 GGH_HUMAN NLDGISHAPNAVK 640 158 158 GGH_HUMAN YYIAASYVK 643 158 0 GRP78_HUMAN TWNDPSVQQDIK 664 158 90 GRP78_HUMAN VYEGERPLTK 665 158 68 GSLG1_HUMAN IIIQESALDYR 666 158 158 GSLG1_HUMAN LDPALQDK 667 158 0 GSLG1_HUMAN LIAQDYK 668 158 0 GSLG1_HUMAN NDINILK 669 158 0 GSTP1_HUMAN ALPGQLKPFETLLSQNQGGK 672 123 123 GSTP1_HUMAN YISLIYTNYEAGK 675 123 0 HPSE_HUMAN LPYPFSNK 714 49 0 HPSE_HUMAN SVQLNGLTLK 715 49 49 HPT_HUMAN VGYVSGWGR 719 158 0 HPT_HUMAN VTSIQDWVQK 720 158 158 HS90A_HUMAN SLTNDWEDHLAVK 724 32 32 HS90B_HUMAN ADHGEPIGR 726 121 0 HS90B_HUMAN IDIIPNPQER 728 121 121 HS90B_HUMAN NPDDITQEEYGEFYK 730 121 0 HSPB1_HUMAN DGVVEITGK 732 30 0 HSPB1_HUMAN GPSWDPFR 733 30 30 HTRA1_HUMAN LHRPPVIVLQR 741 40 40 HTRA1_HUMAN LPVLLLGR 742 40 0 HTRA1_HUMAN VTAGISFAIPSDK 744 40 0 HXK1_HUMAN FLLSESGSGK 747 117 17 HXK1_HUMAN LVDEYSLNAGK 749 117 47 HXK1_HUMAN SANLVAATLGAILNR 750 117 53 HYOU1_HUMAN FPEHELTFDPQR 757 156 0 HYOU1_HUMAN LPATEKPVLLSK 760 156 156 IBP2_HUMAN AEVLFR 762 158 0 IBP2_HUMAN ELAVFR 763 158 158 IBP2_HUMAN LIQGAPTIR 765 158 0 IBP3_HUMAN FHPLHSK 768 158 0 IBP3_HUMAN FLNVLSPR 769 158 0 IBP3_HUMAN YGQPLPGYTTK 771 158 158 ICAM1_HUMAN ASVSVTAEDEGTQR 772 114 0 ICAM1_HUMAN VELAPLPSWQPVGK 776 114 114 ICAM3_HUMAN IALETSLSK 780 158 0 ICAM3_HUMAN TFVLPVTPPR 783 158 158 IF4A1_HUMAN GYDVIAQAQSGTGK 792 58 0 IF4A1_HUMAN VLITTDLLAR 796 58 58 IGF1_HUMAN EGTEASLQIR 797 40 0 IGF1_HUMAN ISSLPTQLFK 798 40 40 IL18_HUMAN SDIIFFQR 807 45 45 IL18_HUMAN SVPGHDNK 808 45 0 ILEU_HUMAN EATTNAPFR 824 88 13 ILEU_HUMAN TYNFLPEFLVSTQK 828 88 75 ILK_HUMAN HSGIDFK 830 90 15 ILK_HUMAN QLNFLTK 832 90 75 ILK_HUMAN WQGNDIVVK 833 90 0 INHBA_HUMAN AEVWLFLK 834 32 0 INHBA_HUMAN EGSDLSVVER 835 32 32 ISLR_HUMAN ALPGTPVASSQPR 839 158 0 ISLR_HUMAN EVPLLQSLWLAHNEIR 840 158 0 ISLR_HUMAN LPGLPEGAFR 841 158 158 ITA5_HUMAN SLQWFGATVR 846 114 114 ITA5_HUMAN SSASSGPQILK 847 114 0 K1C18_HUMAN LASYLDR 861 72 0 K1C18_HUMAN LQLETEIEALK 862 72 72 K1C18_HUMAN VVSETNDTK 863 72 0 K1C19_HUMAN FGAQLAHIQALISGIEAQLGDVR 865 158 158 K1C19_HUMAN FGPGVAFR 866 158 0 KIT_HUMAN QATLTISSAR 876 158 158 KIT_HUMAN YVSELHLTR 878 158 0 KLK14_HUMAN VLGSGTWPSAPK 889 27 27 KLK14_HUMAN VSGWGTISSPIAR 890 27 0 KPYM_HUMAN APIIAVTR 899 158 13 KPYM_HUMAN LDIDSPPITAR 903 158 145 LAMB2_HUMAN IQGTLQPHAR 910 69 0 LAMB2_HUMAN SLADVDAILAR 911 69 31 LAMB2_HUMAN VLELSIPASAEQIQHLAGAIAER 913 69 38 LDHA_HUMAN FIIPNVVK 915 157 0 LDHA_HUMAN LVIITAGAR 917 157 157 LDHB_HUMAN FIIPQIVK 920 158 157 LDHB_HUMAN GLTSVINQK 921 158 1 LEG1_HUMAN GEVAPDAK 925 146 0 LEG1_HUMAN LPDGYEFK 926 146 0 LEG1_HUMAN SFVLNLGK 927 146 146 LG3BP_HUMAN ASHEEVEGLVEK 938 158 158 LG3BP_HUMAN VEIFYR 941 158 0 LG3BP_HUMAN YSSDYFQAPSDYR 942 158 0 LRP1_HUMAN TVLWPNGLSLDIPAGR 959 158 158 LRP1_HUMAN VFFTDYGQIPK 960 158 0 LUM_HUMAN NIPTVNENLENYYLEVNQLEK 962 158 158 LUM_HUMAN SLEDLQLTHNK 964 158 0 LYOX_HUMAN HWFQAGYSTSR 975 121 0 LYOX_HUMAN TPILLIR 977 121 121 MASP1_HUMAN APGELEHGLITFSTR 991 158 151 MASP1_HUMAN TGVITSPDFPNPYPK 994 158 7 MDHC_HUMAN LGVTANDVK 997 130 130 MDHC_HUMAN VLVTGAAGQIAYSLLYSIGNGSVFGK 999 130 0 MDHM_HUMAN VDFPQDQLTALTGR 1002 158 158 MDHM_HUMAN VSSFEEK 1004 158 0 MMP12_HUMAN FLLILLLQATASGALPLNSSTSLEK 1021 158 158 MMP12_HUMAN GIQSLYGDPK 1022 158 0 MMP12_HUMAN IDAVFYSK 1023 158 0 MMP2_HUMAN AFQVWSDVTPLR 1031 153 152 MMP2_HUMAN IIGYTPDLDPETVDDAFAR 1033 153 1 MMP7_HUMAN LSQDDIK 1042 102 102 MMP7_HUMAN NANSLEAK 1043 102 0 MMP9_HUMAN AFALWSAVTPLTFTR 1044 158 50 MMP9_HUMAN FQTFEGDLK 1046 158 108 MMP9_HUMAN SLGPALLLLQK 1048 158 0 MPRI_HUMAN GHQAFDVGQPR 1055 158 23 MPRI_HUMAN TYHSVGDSVLR 1056 158 4 MPRI_HUMAN VPIDGPPIDIGR 1057 158 131 NCF4_HUMAN AEALFDFTGNSK 1095 138 43 NCF4_HUMAN DAEGDLVR 1096 138 0 NCF4_HUMAN DIAVEEDLSSTPLLK 1097 138 0 NCF4_HUMAN GATGIFPLSFVK 1098 138 95 NDKB_HUMAN DRPFFPGLVK 1105 24 0 NDKB_HUMAN NIIHGSDSVK 1107 24 24 NRP1_HUMAN FVSDYETHGAGFSIR 1149 158 0 NRP1_HUMAN FVTAVGTQGAISK 1150 158 158 NRP1_HUMAN SFEGNNNYDTPELR 1152 158 0 OSTP_HUMAN AIPVAQDLNAPSDWDSR 1156 108 108 OSTP_HUMAN DSYETSQLDDQSAETHSHK 1157 108 0 OSTP_HUMAN YPDAVATWLNPDPSQK 1160 108 0 PCBP2_HUMAN IANPVEGSTDR 1189 52 0 PCBP2_HUMAN IITLAGPTNAIFK 1190 52 52 PCYOX_HUMAN IAIIGAGIGGTSAAYYLR 1207 37 0 PCYOX_HUMAN IFSQETLTK 1208 37 37 PCYOX_HUMAN TLLETLQK 1209 37 0 PDGFB_HUMAN SFDDLQR 1216 111 99 PDGFB_HUMAN SHSGGELESLAR 1217 111 12 PDIA3_HUMAN ELSDFISYLQR 1225 129 129 PDIA3_HUMAN SEPIPESNDGPVK 1227 129 0 PDIA4_HUMAN FDVSGYPTIK 1231 81 81 PDIA4_HUMAN FHHTFSTEIAK 1232 81 0 PECA1_HUMAN SELVTVTESFSTPK 1241 77 0 PECA1_HUMAN STESYFIPEVR 1242 77 77 PEDF_HUMAN LQSLFDSPDFSK 1246 158 0 PEDF_HUMAN TVQAVLTVPK 1248 158 158 PGAM1_HUMAN HGESAWNLENR 1259 14 14 PLIN2_HUMAN DAVTTTVTGAK 1264 138 0 PLIN2_HUMAN EVSDSLLTSSK 1265 138 138 PLSL_HUMAN IGNFSTDIK 1284 158 0 PLSL_HUMAN ISFDEFIK 1285 158 158 PLX63_HUMAN ELPVPIYVTQGEAQR 1294 77 0 PLX63_HUMAN GPVDAVTGK 1296 77 77 PLXC1_HUMAN FWVNILK 1299 158 0 PLXC1_HUMAN LNTIGHYEISNGSTIK 1300 158 158 POSTN_HUMAN GFEPGVTNILK 1302 158 158 POSTN_HUMAN IIDGVPVEITEK 1303 158 0 POSTN_HUMAN IIHGNQIATNGVVHVIDR 1304 158 0 PPIB_HUMAN VIFGLFGK 1319 158 0 PPIB_HUMAN VYFDLR 1320 158 158 PRDX1_HUMAN IGHPAPNFK 1325 158 116 PRDX1_HUMAN QITVNDLPVGR 1328 158 42 PROF1_HUMAN STGGAPTFNVTVTK 1338 158 157 PROF1_HUMAN TFVNITPAEVGVLVGK 1339 158 1 PRS6A_HUMAN VDILDPALLR 1349 13 13 PTGIS_HUMAN DPEIYTDPEVFK 1352 158 0 PTGIS_HUMAN LLLFPFLSPQR 1357 158 158 PTPA_HUMAN FGSLLPIHPVTSG 1361 103 103 PTPA_HUMAN TGPFAEHSNQLWNISAVPSWSK 1363 103 0 PTPA_HUMAN VDDQIAIVFK 1364 103 0 PTPA_HUMAN WIDETPPVDQPSR 1365 103 0 PTPRJ_HUMAN AVSISPTNVILTWK 1372 158 0 PTPRJ_HUMAN VITEPIPVSDLR 1374 158 158 PVR_HUMAN SVDIWLR 1379 158 158 PVR_HUMAN VLAKPQNTAEVQK 1380 158 0 RAB32_HUMAN VHLPNGSPIPAVLLANK 1384 22 0 RAB32_HUMAN VLVIGELGVGK 1385 22 22 RAN_HUMAN FNVWDTAGQEK 1391 116 2 RAN_HUMAN LVLVGDGGTGK 1392 116 114 RAN_HUMAN NVPNWHR 1393 116 0 RAP2B_HUMAN EVSYGEGK 1395 145 0 RAP2B_HUMAN VDLEGER 1397 145 145 S10A1_HUMAN DVDAVDK 1408 128 128 S10A1_HUMAN ELLQTELSGFLDAQK 1409 128 0 S10A6_HUMAN ELTIGSK 1411 154 154 S10A6_HUMAN LQDAEIAR 1412 154 0 SAA_HUMAN EANYIGSDK 1414 143 0 SAA_HUMAN SFFSFLGEAFDGAR 1416 143 143 SCF_HUMAN LFTPEEFFR 1418 143 143 SCF_HUMAN LVANLPK 1419 143 0 SEM3G_HUMAN DYPDEVLQFAR 1426 155 0 SEM3G_HUMAN LFLGGLDALYSLR 1428 155 155 SIAL_HUMAN AYEDEYSYFK 1449 19 19 SIAL_HUMAN TTSPPFGK 1452 19 0 SODM_HUMAN GDVTAQIALQPALK 1460 154 151 SODM_HUMAN NVRPDYLK 1462 154 3 SPON2_HUMAN WSQTAFPK 1478 63 0 SPON2_HUMAN YSITFTGK 1479 63 63 STAT1_HUMAN TELISVSEVHPSR 1494 38 29 STAT1_HUMAN YTYEHDPITK 1496 38 9 TBA1B_HUMAN AVFVDLEPTVIDEVR 1519 119 119 TBA1B_HUMAN EIIDLVLDR 1520 119 0 TBB3_HUMAN ISVYYNEASSHK 1533 158 158 TBB3_HUMAN YLTVATVFR 1535 158 0 TCPA_HUMAN IHPTSVISGYR 1540 158 4 TCPA_HUMAN SSLGPVGLDK 1542 158 154 TCPQ_HUMAN DIDEVSSLLR 1550 48 0 TCPQ_HUMAN NVGLDIEAEVPAVK 1553 48 48 TCPZ_HUMAN GIDPFSLDALSK 1557 6 6 TCPZ_HUMAN GLVLDHGAR 1558 6 0 TENA_HUMAN GLEPGQEYNVLLTAEK 1570 140 140 TENA_HUMAN TVSGNTVEYALTDLEPATEYTLR 1572 140 0 TENX_HUMAN DAQGQPQAVPVSGDLR 1574 158 158 TENX_HUMAN YEVTVVSVR 1578 158 0 TERA_HUMAN GILLYGPPGTGK 1579 106 94 TERA_HUMAN LDQLIYIPLPDEK 1582 106 12 TETN_HUMAN GGTLSTPQTGSENDALYEYLR 1588 158 118 TETN_HUMAN LDTLAQEVALLK 1589 158 40 TFR1_HUMAN LTVSNVLK 1598 157 0 TFR1_HUMAN SSGLPNIPVQTISR 1600 157 157 TIMP1_HUMAN GFQALGDAADIR 1610 151 151 TIMP1_HUMAN SEEFLIAGK 1611 151 0 TNF12_HUMAN AAPFLTYFGLFQVH 1621 156 156 TNF12_HUMAN INSSSPLR 1622 156 0 TPIS_HUMAN VVFEQTK 1634 157 157 TPIS_HUMAN VVLAYEPVWAIGTGK 1635 157 0 TRFL_HUMAN FQLFGSPSGQK 1637 48 22 TRFL_HUMAN LRPVAAEVYGTER 1638 48 4 TRFL_HUMAN VPSHAVVAR 1639 48 5 TRFL_HUMAN YYGYTGAFR 1640 48 17 TSP1_HUMAN GFLLLASLR 1644 158 6 TSP1_HUMAN GTSQNDPNWVVR 1645 158 152 TTHY_HUMAN TSESGELHGLTTEEEFVEGIYK 1646 27 27 TTHY_HUMAN VEIDTK 1647 27 0 TYPH_HUMAN ALQEALVLSDR 1648 59 0 TYPH_HUMAN TLVGVGASLGLR 1651 59 59 UGGG1_HUMAN DLSQNFPTK 1653 58 58 UGGG1_HUMAN FTILDSQGK 1654 58 0 UGPA_HUMAN LVEIAQVPK 1669 97 97 UGPA_HUMAN NENTFLDLTVQQIEHLNK 1670 97 0 VA0D1_HUMAN LLFEGAGSNPGDK 1679 13 13 VA0D1_HUMAN NVADYYPEYK 1681 13 0 VEGFC_HUMAN DLEEQLR 1689 21 21 VEGFC_HUMAN EAPAAAAAFESGLDLSDAEPDAGEA 1690 21 0 TAYASK VEGFC_HUMAN FAAAHYNTEILK 1692 21 0 VEGFC_HUMAN NQPLNPGK 1693 21 0 VTNC_HUMAN AVRPGYPK 1705 145 0 VTNC_HUMAN DVWGIEGPIDAAFTR 1707 145 145 ZA2G_HUMAN EIPAWVPFDPAAQITK 1724 158 158 ZA2G_HUMAN WEAEPVYVQR 1725 158 0 Exemplary Biomarker Protein

The following 36 proteins were identified as biomarker candidates in a large-scale experiment of 72 lung cancer samples and 71 benign lung nodule samples using the MS-LC-SRM-MS system described herein.

TABLE 4 Co- Co- efficient efficient Predicted Protein Official Cooper- Co- SEQ (Discovery) (Final) Concen- (Unit- Gene ative Partial efficient ID alpha = alpha = Tissue traion Category Prot) Name Score AUC CV Transition NO: 36.16 26.25 Candiate (ng/ml) Classifier TSP1_ THBS1 1.8 0.25 0.24 GFLLLASLR_ 1644 0.53 0.44 510 HUMAN 495.31_ 559.40 Classifier COIA1_ COL18A1 3.7 0.16 0.25 AVGLAGTFR_ 332 -1.56 -0.91 35 HUMAN 446.26_ 721.40 Classifier ISLR_ ISLR 1.4 0.32 0.25 ALPGTPVAS 839 1.40 0.83 — HUMAN SQPR_ 640.85_ 841.50 Classifier TETN_ CLEC3B 2.5 0.26 0.26 LDTLAQEVAL 1589 -1.79 -1.02 58000 HUMAN LK_ 657.39_ 330.20 Classifier FRIL_ FTL 2.8 0.31 0.26 LFFPAGLG 596 0.36 0.14 Secreted 12 HUMAN EYLFER_ Epi, 804.40_ Endo 913.40 Classifier GRP78_ HSPA5 1.4 0.27 0.27 TWNDPSVQ 664 1.41 0.55 Secreted 100 HUMAN QDIK_ Epi, 715.85_ Endo 260.20 Classifier ALDOA_ ALDOQ 1.3 0.26 0.28 ALQASLK_ 58 -0.80 -0.26 Secreted 250 HUMAN 401.25_ Epi, 617.40 Classifier BGH3_ TGFBI 1.8 0.21 0.28 LTLLAPLNSV 139 1.73 0.54 Epi 1.40 HUMAN FK_ 658.40_ 804.50 Classifier LG3BP_ LGALS3BP 4.3 0.29 0.29 VEIFYR_ 941 -0.58 -0.21 Secreted 440 HUMAN 413.73_ 598.30 Classifier LRP1_ LRP1 4.0 0.13 0.32 TVLWPNGLS 959 -1.59 -0.83 Epi 20 HUMAN LDIPAGR_ 855.00_ 400.20 Classifier FIBA_ FGA 1.1 0.31 0.35 NSLFEYQK_ 556 0.31 0.13 130000 HUMAN 514.76_ 714.30 Classifier PRDX1_ PRDX1 1.5 0.32 0.37 QITVNDLPVG_ 1328 -0.34 -0.26 Epi 60 HUMAN R_606.30_ 428.30 Classifier GSLG1_ GLG1 1.2 0.34 0.45 IIIQESALDYR_ 666 -0.70 -0.44 Epi, — HUMAN 660.86_ Endo 338.20 Robust KIT_ KIT 1.4 0.33 0.46 8.2 HUMAN Robust CD14_ CD14 4.0 0.33 0.48 Epi 420 HUMAN Robust EF1A1_ EF1A1 1.2 0.32 0.56 Secreted, 61 HUMAN Epi Robust TENX_ TNXB 1.1 0.30 0.56 Endo 70 HUMAN Robust AIFM1_ AIFM1 1.4 0.32 0.70 Epi, 1.4 HUMAN Endo Robust GGH_ GGH 1.3 0.32 0.81 250 HUMAN Robust IBP3_ IGFBP3 3.4 0.32 1.82 5700 HUMAN Robust ENPL_ HSP90B1 1.1 0.29 5.90 Secreted, 88 HUMAN Epi, Endo Non- ERO1A_ ERO1L 6.2 Secreted, — Robust HUMAN Epi, Endo Non- 6PGD_ PGD 4.3 Epi, 29 Robust HUMAN Endo Non- ICAM1_ ICAM1_ 3.9 71 Robust HUMAN Non- PTPA_ PPP2R4 2.1 Endo 3.3 Robust HUMAN Non- NCF4_ NCF4 2.0 Endo — Robust HUMAN Non- SEM3G_ SEM3G 1.9 — Robust HUMAN Non- 1433T_ YWHAQ 1.5 Epi 180 Robust HUMAN Non- RAP2B_ RAP2B 1.5 Epi — Robust HUMAN Non- MMP9_ MMP9 1.4 28 Robust HUMAN Non- FOLH1_ FOLH1 1.3 — Robust HUMAN Non- GSTP1_ GSTP1 1.3 Endo 32 Robust HUMAN Non- EF2_ EEF2 1.3 Secreted, 30 Robust HUMAN Epi Non- RAN_ RAN 1.2 Secreted, 4.6 Robust HUMAN Epi Non- SODM_ SOD2 1.2 Secreted 7.1 Robust HUMAN Non- DSG2_ DSG2 1.1 Endo 2.7 Robust HUMAN

LENGTHY TABLES The patent contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US10534002B2). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3). 

What is claimed is:
 1. A composition comprising isolated synthetic peptides said composition comprising ALQASALK (SEQ ID NO: 58), GFLLLASLR (SEQ ID NO: 1644), LGGPEAGLGEYLFER (SEQ ID NO: 596), VEIFYR (SEQ ID NO: 941), INPASLDK (SEQ ID NO: 153) and TSYQVYSK (SEQ ID NO: 155).
 2. A composition comprising at least 10 isolated synthetic peptides said composition comprising at least 2 isolated synthetic peptides of each TSP1, ALDOA, FRIL, LG3BP, and C163A, and wherein the at least two isolated synthetic peptides are selected from ADDGRPFPQVIK (SEQ ID NO: 57), ALQASALK (SEQ ID NO: 58), QLLLTADDR (SEQ ID NO: 60), ALFQDIK (SEQ ID NO: 593), DDV ALEGVSHFFR (SEQ ID NO: 594), KPAEDEWGK (SEQ ID NO: 595), LGGPEAGLGEYLFER(SEQ IDNO: 596), LNQALLDLHALGSAR (SEQ ID NO: 597), MGDHLTNLHR (SEQ ID NO: 598), ASHEEVEGL VEK (SEQ ID NO: 938), STHTLDLSR (SEQ ID NO: 940), VEIFYR (SEQ ID NO: 941), YSSDYFQAPSDYR (SEQ ID NO: 942), FQDLVDA VR (SEQ ID NO: 1641), FTGSQPFGQGVEHATANK (SEQ ID NO: 1642), FVFGTIPEDILR (SEQ ID NO: 1643), GFLLLASLR (SEQ ID NO: 1644), INPASLDK (SEQ ID NO: 153), LEVFYNGAWGTVGK (SEQ ID NO: 154), TSYQVYSK (SEQ ID NO: 155), and GTSQNDPNWVVR (SEQ ID NO: 1645).
 3. The composition of claim 2, wherein at least one of the isolated synthetic peptides is isotopically labeled.
 4. The composition of claim 2, wherein the measured quantity of each of the at least 2 isolated synthetic peptides of each TSP1, ALDOA, FRIL, LG3BP, and C163A is known.
 5. The composition of claim 2, further comprising one or more polar solvents.
 6. The composition of claim 2, further comprising an additional five isolated synthetic peptides of at least one protein selected from LRP1, BGH3, COIA1, TETN, TSP1, ALDOA, GRP78, ISLR, FRIL, LG3BP, PRDX1, FIBA, KIT, GGH, C163A and GSLGI.
 7. The composition of claim 2, further comprising an additional five isolated synthetic peptides of at least one protein selected from APOE, BASP1, CD14, FOXA2 or HSPB
 1. 8. A composition comprising at least 25 isolated synthetic peptides said composition comprising at least five isolated synthetic peptides of each TSP1, ALDOA, FRIL, LG3BP, and C163A wherein the at least five isolated synthetic peptides are selected from ADDGRPFPQVIK (SEQ ID NO: 57), ALQASALK (SEQ ID NO: 58), QLLLTADDR (SEQ ID NO: 60), ALFQDIK (SEQ ID NO: 593), DDV ALEGVSHFFR (SEQ ID NO: 594), KPAEDEWGK (SEQ IDNO: 595), LGGPEAGLGEYLFER(SEQ IDNO: 596), LNQALLDLHALGSAR (SEQ ID NO: 597), MGDHLTNLHR (SEQ ID NO: 598), ASHEEVEGLVEK (SEQ ID NO: 938), STHTLDLSR (SEQ ID NO: 940), VEIFYR (SEQ ID NO: 941), YSSDYFQAPSDYR (SEQ ID NO: 942), FQDLVDAVR (SEQ ID NO: 1641), FTGSQPFGQGVEHATANK (SEQ ID NO: 1642), FVFGTIPEDILR (SEQ ID NO: 1643), GFLLLASLR (SEQ ID NO: 1644), INPASLDK (SEQ ID NO: 153), LEVFYNGAWGTVGK (SEQ ID NO: 154), TSYQVYSK (SEQ ID NO: 155), and GTSQNDPNWVVR (SEQ ID NO: 1645).
 9. The composition of claim 8, wherein the at least five isolated synthetic peptides are selected from GFLLLASLR (SEQ ID NO: 1644), LGGPEAGLGEYLFER (SEQ ID NO: 596), ALQASALK (SEQ ID NO: 58), VEIFYR (SEQ ID NO: 941), INPASLDK (SEQ ID NO: 153), LEVFYNGAWGTVGK (SEQ ID NO: 154), and TSYQVYSK (SEQ ID NO: 155).
 10. A method comprising analyzing the composition of claim 8 using mass spectrometry.
 11. The method of claim 10, wherein the method uses selected reaction monitoring mass spectrometry.
 12. A method comprising analyzing the composition of claim 8 using Enzyme-Linked Immunosorbent Assay (ELISA). 